Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter peptides, and methods of identifying modulators of the transporter peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of transporter proteins that are related to the chloride intracellular channel subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides a novel human isoform of an ion channel protein and nucleic acid molecules encoding the novel isoform, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0002] Transporters

[0003] Transporter proteins regulate many different functions of a cell, including cell proliferation, differentiation, and signaling processes, by regulating the flow of molecules such as ions and macromolecules, into and out of cells. Transporters are found in the plasma membranes of virtually every cell in eukaryotic organisms. Transporters mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of molecules and ion across cell membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, transporters, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

[0004] Transporters are generally classified by structure and the type of mode of action. In addition, transporters are sometimes classified by the molecule type that is transported, for example, sugar transporters, chlorine channels, potassium channels, etc. There may be many classes of channels for transporting a single type of molecule (a detailed review of channel types can be found at Alexander, S. P. H. and J. A. Peters: Receptor and transporter nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 (1997) and http://www-biology.ucsd.edu/˜msaier/transport/titlepage2.html.

[0005] The following general classification scheme is known in the art and is followed in the present discoveries.

[0006] Channel-type transporters. Transmembrane channel proteins of this class are ubiquitously found in the membranes of all types of organisms from bacteria to higher eukaryotes. Transport systems of this type catalyze facilitated diffusion (by an energy-independent process) by passage through a transmembrane aqueous pore or channel without evidence for a carrier-mediated mechanism. These channel proteins usually consist largely of a-helical spanners, although b-strands may also be present and may even comprise the channel. However, outer membrane porin-type channel proteins are excluded from this class and are instead included in class 9.

[0007] Carrier-type transporters. Transport systems are included in this class if they utilize a carrier-mediated process to catalyze uniport (a single species is transported by facilitated diffusion), antiport (two or more species are transported in opposite directions in a tightly coupled process, not coupled to a direct form of energy other than chemiosmotic energy) and/or symport (two or more species are transported together in the same direction in a tightly coupled process, not coupled to a direct form of energy other than chemiosmotic energy).

[0008] Pyrophosphate bond hydrolysis-driven active transporters. Transport systems are included in this class if they hydrolyze pyrophosphate or the terminal pyrophosphate bond in ATP or another nucleoside triphosphate to drive the active uptake and/or extrusion of a solute or solutes. The transport protein may or may not be transiently phosphorylated, but the substrate is not phosphorylated.

[0009] PEP-dependent, phosphoryl transfer-driven group translocators. Transport systems of the bacterial phosphoenolpyruvate:sugar phosphotransferase system are included in this class. The product of the reaction, derived from extracellular sugar, is a cytoplasmic sugar-phosphate.

[0010] Decarboxylation-driven active transporters. Transport systems that drive solute (e.g., ion) uptake or extrusion by decarboxylation of a cytoplasmic substrate are included in this class.

[0011] Oxidoreduction-driven active transporters. Transport systems that drive transport of a solute (e.g., an ion) energized by the flow of electrons from a reduced substrate to an oxidized substrate are included in this class.

[0012] Light-driven active transporters. Transport systems that utilize light energy to drive transport of a solute (e.g., an ion) are included in this class.

[0013] Mechanically-driven active transporters. Transport systems are included in this class if they drive movement of a cell or organelle by allowing the flow of ions (or other solutes) through the membrane down their electrochemical gradients.

[0014] Outer-membrane porins (of b-structure). These proteins form transmembrane pores or channels that usually allow the energy independent passage of solutes across a membrane. The transmembrane portions of these proteins consist exclusively of b-strands that form a b-barrel. These porin-type proteins are found in the outer membranes of Gram-negative bacteria, mitochondria and eukaryotic plastids.

[0015] Methyltransferase-driven active transporters. A single characterized protein currently falls into this category, the Na+-transporting methyltetrahydromethanopterin:coenzyme M methyltransferase.

[0016] Non-ribosome-synthesized channel-forming peptides or peptide-like molecules. These molecules, usually chains of L- and D-amino acids as well as other small molecular building blocks such as lactate, form oligomeric transmembrane ion channels. Voltage may induce channel formation by promoting assembly of the transmembrane channel. These peptides are often made by bacteria and fungi as agents of biological warfare.

[0017] Non-Proteinaceous Transport Complexes. Ion conducting substances in biological membranes that do not consist of or are not derived from proteins or peptides fall into this category.

[0018] Functionally characterized transporters for which sequence data are lacking. Transporters of particular physiological significance will be included in this category even though a family assignment cannot be made.

[0019] Putative transporters in which no family member is an established transporter. Putative transport protein families are grouped under this number and will either be classified elsewhere when the transport function of a member becomes established, or will be eliminated from the TC classification system if the proposed transport function is disproven. These families include a member or members for which a transport function has been suggested, but evidence for such a function is not yet compelling.

[0020] Auxiliary transport proteins. Proteins that in some way facilitate transport across one or more biological membranes but do not themselves participate directly in transport are included in this class. These proteins always function in conjunction with one or more transport proteins. They may provide a function connected with energy coupling to transport, play a structural role in complex formation or serve a regulatory function.

[0021] Transporters of unknown classification. Transport protein families of unknown classification are grouped under this number and will be classified elsewhere when the transport process and energy coupling mechanism are characterized. These families include at least one member for which a transport function has been established, but either the mode of transport or the energy coupling mechanism is not known.

[0022] Ion Channels

[0023] An important type of transporter is the ion channel. Ion channels regulate many different cell proliferation, differentiation, and signaling processes by regulating the flow of ions into and out of cells. Ion channels are found in the plasma membranes of virtually every cell in eukaryotic organisms. Ion channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ion across epithelial membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, ion channels, such as chloride channels, also regulate organelle pH. For a review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

[0024] Ion channels are generally classified by structure and the type of mode of action. For example, extracellular ligand gated channels (ELGs) are comprised of five polypeptide subunits, with each subunit having 4 membrane spanning domains, and are activated by the binding of an extracellular ligand to the channel. In addition, channels are sometimes classified by the ion type that is transported, for example, chlorine channels, potassium channels, etc. There may be many classes of channels for transporting a single type of ion (a detailed review of channel types can be found at Alexander, S. P. H. and J. A. Peters (1997). Receptor and ion channel nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp. 65-68 and http://www-biology.ucsd.edu/˜msaier/transport/toc.html.

[0025] There are many types of ion channels based on structure. For example, many ion channels fall within one of the following groups: extracellular ligand-gated channels (ELG), intracellular ligand-gated channels (ILG), inward rectifying channels (INR), intercellular (gap junction) channels, and voltage gated channels (VIC). There are additionally recognized other channel families based on ion-type transported, cellular location and drug sensitivity. Detailed information on each of these, their activity, ligand type, ion type, disease association, drugability, and other information pertinent to the present invention, is well known in the art.

[0026] Extracellular ligand-gated channels, ELGs, are generally comprised of five polypeptide subunits, Unwin, N. (1993), Cell 72: 31-41; Unwin, N. (1995), Nature 373: 37-43; Hucho, F., et al., (1996) J. Neurochem. 66: 1781-1792; Hucho, F., et al., (1996) Eur. J. Biochem. 239: 539-557; Alexander, S. P. H. and J. A. Peters (1997), Trends Pharmacol. Sci., Elsevier, pp. 4-6; 36-40; 42-44; and Xue, H. (1998) J. Mol. Evol. 47: 323-333. Each subunit has 4 membrane spanning regions: this serves as a means of identifying other members of the ELG family of proteins. ELG bind a ligand and in response modulate the flow of ions. Examples of ELG include most members of the neurotransmitter-receptor family of proteins, e.g., GABAI receptors. Other members of this family of ion channels include glycine receptors, ryandyne receptors, and ligand gated calcium channels.

[0027] The Voltage-Gated Ion Channel (VIC) Superfamily

[0028] Proteins of the VIC family are ion-selective channel proteins found in a wide range of bacteria, archaea and eukaryotes Hille, B. (1992), Chapter 9: Structure of channel proteins; Chapter 20: Evolution and diversity. In: Ionic Channels of Excitable Membranes, 2nd Ed., Sinaur Assoc. Inc., Pubs., Sunderland, Massachusetts; Sigworth, F. J. (1993), Quart. Rev. Biophys. 27: 1-40; Salkoff, L. and T. Jegla (1995), Neuron 15: 489-492; Alexander, S. P. H. et al., (1997), Trends Pharmacol. Sci., Elsevier, pp. 76-84; Jan, L. Y. et al., (1997), Annu. Rev. Neurosci. 20: 91-123; Doyle, D. A, et al., (1998) Science 280: 69-77; Terlau, H. and W. Stühmer (1998), Naturwissenschaften 85: 437-444. They are often homo- or heterooligomeric structures with several dissimilar subunits (e.g., a1-a2-d-b Ca²⁺ channels, ab₁b₂ Na⁺ channels or (a)₄-b K⁺ channels), but the channel and the primary receptor is usually associated with the a (or a1) subunit. Functionally characterized members are specific for K⁺, Na⁺ or Ca²⁺. The K⁺ channels usually consist of homotetrameric structures with each a-subunit possessing six transmembrane spanners (TMSs). The a1 and a subunits of the Ca²⁺ and Na⁺ channels, respectively, are about four times as large and possess 4 units, each with 6 TMSs separated by a hydrophilic loop, for a total of 24 TMSs. These large channel proteins form heterotetra-unit structures equivalent to the homotetrameric structures of most K⁺ channels. All four units of the Ca²⁺ and Na⁺ channels are homologous to the single unit in the homotetrameric K⁺ channels. Ion flux via the eukaryotic channels is generally controlled by the transmembrane electrical potential (hence the designation, voltage-sensitive) although some are controlled by ligand or receptor binding.

[0029] Several putative K⁺-selective channel proteins of the VIC family have been identified in prokaryotes. The structure of one of them, the KcsA K⁺ channel of Streptomyces lividans, has been solved to 3.2 Å resolution. The protein possesses four identical subunits, each with two transmembrane helices, arranged in the shape of an inverted teepee or cone. The cone cradles the “selectivity filter” P domain in its outer end. The narrow selectivity filter is only 12 Å long, whereas the remainder of the channel is wider and lined with hydrophobic residues. A large water-filled cavity and helix dipoles stabilize K⁺ in the pore. The selectivity filter has two bound K⁺ ions about 7.5 Å apart from each other. Ion conduction is proposed to result from a balance of electrostatic attractive and repulsive forces.

[0030] In eukaryotes, each VIC family channel type has several subtypes based on pharmacological and electrophysiological data. Thus, there are five types of Ca²⁺ channels (L, N, P, Q and T). There are at least ten types of K⁺ channels, each responding in different ways to different stimuli: voltage-sensitive [Ka, Kv, Kvr, Kvs and Ksr], Ca2+-sensitive [BK_(Ca), IK_(Ca) and SK_(Ca)] and receptor-coupled [K_(M) and K_(ACh)]. There are at least six types of Na⁺ channels (I, II, III, μ1, H1 and PN3). Tetrameric channels from both prokaryotic and eukaryotic organisms are known in which each a-subunit possesses 2 TMSs rather than 6, and these two TMSs are homologous to TMSs 5 and 6 of the six TMS unit found in the voltage-sensitive channel proteins. KcsA of S. lividans is an example of such a 2 TMS channel protein. These channels may include the K_(Na) (Na⁺-activated) and K_(Vol) (cell volume-sensitive) K⁺ channels, as well as distantly related channels such as the Tok1 K⁺ channel of yeast, the TWIK-1 inward rectifier K⁺ channel of the mouse and the TREK-1 K⁺ channel of the mouse. Because of insufficient sequence similarity with proteins of the VIC family, inward rectifier K⁺ IRK channels (ATP-regulated; G-protein-activated) which possess a P domain and two flanking TMSs are placed in a distinct family. However, substantial sequence similarity in the P region suggests that they are homologous. The b, g and d subunits of VIC family members, when present, frequently play regulatory roles in channel activation/deactivation.

[0031] The Epithelial Na⁺ Channel (ENaC) Family

[0032] The ENaC family consists of over twenty-four sequenced proteins (Canessa, C. M., et al., (1994), Nature 367: 463-467, Le, T. and M. H. Saier, Jr. (1996), Mol. Membr. Biol. 13: 149-157; Garty, H. and L. G. Palmer (1997), Physiol. Rev. 77: 359-396; Waldmann, R., et al., (1997), Nature 386: 173-177; Darboux, I., et al., (1998), J. Biol. Chem. 273: 9424-9429; Firsov, D., et al., (1998), EMBO J. 17: 344-352; Horisberger, J. -D. (1998). Curr. Opin. Struc. Biol. 10: 443-449). All are from animals with no recognizable homologues in other eukaryotes or bacteria. The vertebrate ENaC proteins from epithelial cells cluster tightly together on the phylogenetic tree: voltage-insensitive ENaC homologues are also found in the brain. Eleven sequenced C. elegans proteins, including the degenerins, are distantly related to the vertebrate proteins as well as to each other. At least some of these proteins form part of a mechano-transducing complex for touch sensitivity. The homologous Helix aspersa (FMRF-amide)-activated Na⁺ channel is the first peptide neurotransmitter-gated ionotropic receptor to be sequenced.

[0033] Protein members of this family all exhibit the same apparent topology, each with N- and C-termini on the inside of the cell, two amphipathic transmembrane spanning segments, and a large extracellular loop. The extracellular domains contain numerous highly conserved cysteine residues. They are proposed to serve a receptor function.

[0034] Mammalian ENaC is important for the maintenance of Na⁺ balance and the regulation of blood pressure. Three homologous ENaC subunits, alpha, beta, and gamma, have been shown to assemble to form the highly Na⁺-selective channel. The stoichiometry of the three subunits is alpha₂, beta1, gamma1 in a heterotetrameric architecture.

[0035] The Glutamate-Gated Ion Channel (GIC) Family of Neurotransmitter Receptors

[0036] Members of the GIC family are heteropentameric complexes in which each of the 5 subunits is of 800-1000 amino acyl residues in length (Nakanishi, N., et al, (1990), Neuron 5: 569-581; Unwin, N. (1993), Cell 72: 31-41; Alexander, S. P. H. and J. A. Peters (1997) Trends Pharmacol. Sci., Elsevier, pp. 36-40). These subunits may span the membrane three or five times as putative a-helices with the N-termini (the glutamate-binding domains) localized extracellularly and the C-termini localized cytoplasmically. They may be distantly related to the ligand-gated ion channels, and if so, they may possess substantial b-structure in their transmembrane regions. However, homology between these two families cannot be established on the basis of sequence comparisons alone. The subunits fall into six subfamilies: a, b, g, d, e and z.

[0037] The GIC channels are divided into three types: (1) a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-, (2) kainate- and (3) N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Subunits of the AMPA and kainate classes exhibit 35-40% identity with each other while subunits of the NMDA receptors exhibit 22-24% identity with the former subunits. They possess large N-terminal, extracellular glutamate-binding domains that are homologous to the periplasmic glutamine and glutamate receptors of ABC-type uptake permeases of Gram-negative bacteria. All known members of the GIC family are from animals. The different channel (receptor) types exhibit distinct ion selectivities and conductance properties. The NMDA-selective large conductance channels are highly permeable to monovalent cations and Ca²⁺. The AMPA- and kainate-selective ion channels are permeable primarily to monovalent cations with only low permeability to Ca²⁺.

[0038] The Chloride Channel (ClC) Family

[0039] The ClC family is a large family consisting of dozens of sequenced proteins derived from Gram-negative and Gram-positive bacteria, cyanobacteria, archaea, yeast, plants and animals (Steinmeyer, K., et al., (1991), Nature 354: 301-304; Uchida, S., et al., (1993), J. Biol. Chem. 268: 3821-3824; Huang, M. -E., et al., (1994), J. Mol. Biol. 242: 595-598; Kawasaki, M., et al, (1994), Neuron 12: 597-604; Fisher, W. E., et al., (1995), Genomics. 29:598-606; and Foskett, J. K. (1998), Annu. Rev. Physiol. 60: 689-717). These proteins are essentially ubiquitous, although they are not encoded within genomes of Haemophilus influenzae, Mycoplasma genitalium, and Mycoplasma pneumoniae. Sequenced proteins vary in size from 395 amino acyl residues (M. jannaschii) to 988 residues (man). Several organisms contain multiple ClC family paralogues. For example, Synechocystis has two paralogues, one of 451 residues in length and the other of 899 residues. Arabidopsis thaliana has at least four sequenced paralogues, (775-792 residues), humans also have at least five paralogues (820-988 residues), and C. elegans also has at least five (810-950 residues). There are nine known members in mammals, and mutations in three of the corresponding genes cause human diseases. E. coli, Methanococcus jannaschii and Saccharomyces cerevisiae only have one ClC family member each. With the exception of the larger Synechocystis paralogue, all bacterial proteins are small (395-492 residues) while all eukaryotic proteins are larger (687-988 residues). These proteins exhibit 10-12 putative transmembrane a-helical spanners (TMSs) and appear to be present in the membrane as homodimers. While one member of the family, Torpedo ClC-O, has been reported to have two channels, one per subunit, others are believed to have just one.

[0040] All functionally characterized members of the ClC family transport chloride, some in a voltage-regulated process. These channels serve a variety of physiological functions (cell volume regulation; membrane potential stabilization; signal transduction; transepithelial transport, etc.). Different homologues in humans exhibit differing anion selectivities, i.e., ClC4 and ClC5 share a NO₃ ⁻>Cl⁻>Br⁻>I⁻ conductance sequence, while ClC3 has an I⁻>Cl⁻ selectivity. The ClC4 and ClC5 channels and others exhibit outward rectifying currents with currents only at voltages more positive than +20 mV.

[0041] Chloride Intracellular Channel (CLIC)

[0042] The novel human protein, and encoding gene, provided by the present invention is a novel isoform of chloride intracellular channel 5 (CLIC5) (Genbank gi8393147). Specifically, the isoform of the present invention differs from the art-known CLIC5 isoform at the 3′ end. The isoform provided by the present invention, having a novel 3′ end, is supported by EST data (see FIG. 2). Furthermore, the 3′ end of the cDNA molecule of the present invention is intact after the stop codon, and the stop codon and polyA signal are present in the genomic sequence.

[0043] CLIC5 has been isolated from placental microvilli, where it exists as a component of a multimeric complex consisting of actin, ezrin, alpha-actinin, gelsolin, IQGAP1, and other known cytoskeletal proteins. CLIC5 is enriched in placental microvilli compared with the CLIC1 isoform and CLIC5 is associated with the detergent-insoluble cytoskeletal fraction of microvilli. CLIC5 is concentrated within the apical region of the trophoblast. It has been suggested that CLIC5 plays a distinct role in chloride transport compared with the CLIC1 and CLIC4 isoforms, and that CLIC5 interacts with the cortical actin cytoskeleton in polarized epithelial cells (Berryman et al., Mol Biol Cell May 2000; 11(5):1509-21).

[0044] Intracellular voltage gated ion channels reside in the cellular organelles. They regulate membrane potentials of intracellular membranes and plasma membrane. These proteins may be extracted from microsomes and nuclei. Intracellular ion channels belong to the CLIC family of channels. These proteins are represented by several isoforms in mammalian genomes. Higher levels of CLIC expression is observed in the heart, kidney, and skeletal muscle.

[0045] CLICs play an essential role in transepithelial ion transport. Increasing cell volume (osmotic swelling) activates some chloride channels. Intestinal chloride channels can be vital for a number of physiological processes, such as digestion, electrolyte homeostasis and peristalsis. Possible interaction of CLICs with other components of the transepithelial transport machine, such as CFTR or cystic fibrosis transmembrane conductance regulator, may shed light on the mechanisms of diseases associated with impaired water/ion balance. The role of CLICs in acidification of intracellular vesicles may be crucial for virus assembly and propagation.

[0046] Interestingly, the intracellular ion channels, and CLICs in particular, may be essential for cell division and apoptosis (cell death). Although their roles in the cell cycle are not well documented at this time, there is a possibility they can be up-regulated in rapidly dividing cells such as cancer cells. In this case, synthetic CLIC mediators may be delivered to the transformed tissue in order to slow cancerous growth.

[0047] At least three inherited diseases are associated with mutations in chloride channels: myotonia congenita, Dent's disease, and Bartter's syndrome. The sequences provided by the present invention can be used to screen human populations for allele variations and associated disorders. Inhibitors or activators of the protein of the present invention can be used as therapeutic agents to treat diseases of the digestive tract.

[0048] For a further review of chloride intracellular channel, see Li et al., J Biol Chem Sep. 5, 2000; Clarke et al., Am J Physiol Gastrointest Liver Physiol July 2000; 279(1):G132-8; Franco-Obregon et al., Biophys J July 2000; 79(1):202-14; and Waldegger et al., J Am Soc Nephrol July 2000; 11(7):1331-9.

[0049] Animal Inward Rectifier K⁺ Channel (IRK-C) Family

[0050] IRK channels possess the “minimal channel-forming structure” with only a P domain, characteristic of the channel proteins of the VIC family, and two flanking transmembrane spanners (Shuck, M. E., et al., (1994), J. Biol. Chem. 269: 24261-24270; Ashen, M. D., et al., (1995), Am. J. Physiol. 268: H506-H511; Salkoff, L. and T. Jegla (1995), Neuron 15: 489-492; Aguilar-Bryan, L., et al., (1998), Physiol. Rev. 78: 227-245; Ruknudin, A., et al., (1998), J. Biol. Chem. 273: 14165-14171). They may exist in the membrane as homo- or heterooligomers. They have a greater tendency to let K⁺ flow into the cell than out. Voltage-dependence may be regulated by external K⁺, by internal Mg²⁺, by internal ATP and/or by G-proteins. The P domains of IRK channels exhibit limited sequence similarity to those of the VIC family, but this sequence similarity is insufficient to establish homology. Inward rectifiers play a role in setting cellular membrane potentials, and the closing of these channels upon depolarization permits the occurrence of long duration action potentials with a plateau phase. Inward rectifiers lack the intrinsic voltage sensing helices found in VIC family channels. In a few cases, those of Kir1.1a and Kir6.2, for example, direct interaction with a member of the ABC superfamily has been proposed to confer unique functional and regulatory properties to the heteromeric complex, including sensitivity to ATP. The SUR1 sulfonylurea receptor (spQ09428) is the ABC protein that regulates the Kir6.2 channel in response to ATP, and CFTR may regulate Kir1.1a. Mutations in SUR1 are the cause of familial persistent hyperinsulinemic hypoglycemia in infancy (PHHI), an autosomal recessive disorder characterized by unregulated insulin secretion in the pancreas.

[0051] ATP-Gated Cation Channel (ACC) Family

[0052] Members of the ACC family (also called P2X receptors) respond to ATP, a functional neurotransmitter released by exocytosis from many types of neurons (North, R. A. (1996), Curr. Opin. Cell Biol. 8: 474-483; Soto, F., M. Garcia-Guzman and W. Stühmer (1997), J. Membr. Biol. 160: 91-100). They have been placed into seven groups (P2X₁-P2X₇) based on their pharmacological properties. These channels, which function at neuron-neuron and neuron-smooth muscle junctions, may play roles in the control of blood pressure and pain sensation. They may also function in lymphocyte and platelet physiology. They are found only in animals.

[0053] The proteins of the ACC family are quite similar in sequence (>35% identity), but they possess 380-1000 amino acyl residues per subunit with variability in length localized primarily to the C-terminal domains. They possess two transmembrane spanners, one about 30-50 residues from their N-termini, the other near residues 320-340. The extracellular receptor domains between these two spanners (of about 270 residues) are well conserved with numerous conserved glycyl and cysteyl residues. The hydrophilic C-termini vary in length from 25 to 240 residues. They resemble the topologically similar epithelial Na⁺ channel (ENaC) proteins in possessing (a) N- and C-termini localized intracellularly, (b) two putative transmembrane spanners, (c) a large extracellular loop domain, and (d) many conserved extracellular cysteyl residues. ACC family members are, however, not demonstrably homologous with them. ACC channels are probably hetero- or homomultimers and transport small monovalent cations (Me⁺). Some also transport Ca²⁺; a few also transport small metabolites.

[0054] The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca²⁺ Channel (RIR-CaC) Family

[0055] Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate (IP3)-sensitive Ca²⁺-release channels function in the release of Ca²⁺ from intracellular storage sites in animal cells and thereby regulate various Ca²⁺-dependent physiological processes (Hasan, G. et al., (1992) Development 116: 967-975; Michikawa, T., et al., (1994), J. Biol. Chem. 269: 9184-9189; Tunwell, R. E. A., (1996), Biochem. J. 318: 477-487; Lee, A. G. (1996) Biomembranes, Vol. 6, Transmembrane Receptors and Channels (A. G. Lee, ed.), JAI Press, Denver, Colo., pp 291-326; Mikoshiba, K., et al., (1996) J. Biochem. Biomem. 6: 273-289). Ry receptors occur primarily in muscle cell sarcoplasmic reticular (SR) membranes, and IP3 receptors occur primarily in brain cell endoplasmic reticular (ER) membranes where they effect release of Ca²⁺ into the cytoplasm upon activation (opening) of the channel.

[0056] The Ry receptors are activated as a result of the activity of dihydropyridine-sensitive Ca²⁺ channels. The latter are members of the voltage-sensitive ion channel (VIC) family. Dihydropyridine-sensitive channels are present in the T-tubular systems of muscle tissues.

[0057] Ry receptors are homotetrameric complexes with each subunit exhibiting a molecular size of over 500,000 daltons (about 5,000 amino acyl residues). They possess C-terminal domains with six putative transmembrane a -helical spanners (TMSs). Putative pore-forming sequences occur between the fifth and sixth TMSs as suggested for members of the VIC family. The large N-terminal hydrophilic domains and the small C-terminal hydrophilic domains are localized to the cytoplasm. Low resolution 3-dimensional structural data are available. Mammals possess at least three isoforms that probably arose by gene duplication and divergence before divergence of the mammalian species. Homologues are present in humans and Caenorabditis elegans.

[0058] IP3 receptors resemble Ry receptors in many respects. (1) They are homotetrameric complexes with each subunit exhibiting a molecular size of over 300,000 daltons (about 2,700 amino acyl residues). (2) They possess C-terminal channel domains that are homologous to those of the Ry receptors. (3) The channel domains possess six putative TMSs and a putative channel lining region between TMSs 5 and 6. (4) Both the large N-terminal domains and the smaller C-terminal tails face the cytoplasm. (5) They possess covalently linked carbohydrate on extracytoplasmic loops of the channel domains. (6) They have three currently recognized isoforms (types 1, 2, and 3) in mammals which are subject to differential regulation and have different tissue distributions.

[0059] IP₃ receptors possess three domains: N-terminal IP₃-binding domains, central coupling or regulatory domains and C-terminal channel domains. Channels are activated by IP₃ binding, and like the Ry receptors, the activities of the IP₃ receptor channels are regulated by phosphorylation of the regulatory domains, catalyzed by various protein kinases. They predominate in the endoplasmic reticular membranes of various cell types in the brain but have also been found in the plasma membranes of some nerve cells derived from a variety of tissues.

[0060] The channel domains of the Ry and IP₃ receptors comprise a coherent family that in spite of apparent structural similarities, do not show appreciable sequence similarity of the proteins of the VIC family. The Ry receptors and the IP₃ receptors cluster separately on the RIR-CaC family tree. They both have homologues in Drosophila. Based on the phylogenetic tree for the family, the family probably evolved in the following sequence: (1) A gene duplication event occurred that gave rise to Ry and IP₃ receptors in invertebrates. (2) Vertebrates evolved from invertebrates. (3) The three isoforms of each receptor arose as a result of two distinct gene duplication events. (4) These isoforms were transmitted to mammals before divergence of the mammalian species.

[0061] The Organellar Chloride Channel (O-ClC) Family

[0062] Proteins of the O-ClC family are voltage-sensitive chloride channels found in intracellular membranes but not the plasma membranes of animal cells (Landry, D, et al., (1993), J. Biol. Chem. 268: 14948-14955; Valenzuela, Set al., (1997), J. Biol. Chem. 272: 12575-12582; and Duncan, R. R., et al., (1997), J. Biol. Chem. 272: 23880-23886).

[0063] They are found in human nuclear membranes, and the bovine protein targets to the microsomes, but not the plasma membrane, when expressed in Xenopus laevis oocytes. These proteins are thought to function in the regulation of the membrane potential and in transepithelial ion absorption and secretion in the kidney. They possess two putative transmembrane a-helical spanners (TMSs) with cytoplasmic N- and C-termini and a large luminal loop that may be glycosylated. The bovine protein is 437 amino acyl residues in length and has the two putative TMSs at positions 223-239 and 367-385. The human nuclear protein is much smaller (241 residues). A C. elegans homologue is 260 residues long.

[0064] Transporter proteins, particularly members of the chloride intracellular channel subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown transport proteins. The present invention advances the state of the art by providing previously unidentified human transport proteins.

SUMMARY OF THE INVENTION

[0065] The present invention is based in part on the identification of amino acid sequences of a novel human chloride intracellular channel isoform, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, representing a novel isoform, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate ion channel activity in cells and tissues that express the isoform. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain.

DESCRIPTION OF THE FIGURE SHEETS

[0066]FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodes the transporter protein of the present invention. (SEQ ID NO: 1) In addition structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain.

[0067]FIG. 2 provides the predicted amino acid sequence of the transporter of the present invention. (SEQ ID NO: 2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

[0068]FIG. 3 provides genomic sequences that span the gene encoding the transporter protein of the present invention. (SEQ ID NO: 3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in FIG. 3, SNPs were identified at 116 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0069] General Description

[0070] The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a transporter protein or part of a transporter protein and are related to the chloride intracellular channel subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of a novel human chloride intracellular channel isoform (interchangeably referred to herein as the isoform, transporter, or ion channel of the present invention), nucleic acid sequences in the form of transcript/cDNA sequences and genomic sequences that encode this isoform, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the isoform of the present invention.

[0071] In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known transporter proteins of the chloride intracellular channel subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain.. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known chloride intracellular channel family or subfamily of transporter proteins.

[0072] Specific Embodiments

[0073] Peptide Molecules

[0074] The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the transporter family of proteins and are related to the chloride intracellular channel subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the transporter peptides of the present invention, transporter peptides, or peptides/proteins of the present invention.

[0075] The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprising the amino acid sequences of the transporter peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

[0076] As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

[0077] In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

[0078] The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the transporter peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

[0079] The isolated transporter peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. For example, a nucleic acid molecule encoding the transporter peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

[0080] Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO: 2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQ ID NO: 3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

[0081] The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO: 2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQ ID NO: 3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

[0082] The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO: 2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQ ID NO: 3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the transporter peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

[0083] The transporter peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a transporter peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the transporter peptide. “Operatively linked” indicates that the transporter peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the transporter peptide.

[0084] In some uses, the fusion protein does not affect the activity of the transporter peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant transporter peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

[0085] A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A transporter peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the transporter peptide.

[0086] As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

[0087] Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the transporter peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

[0088] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0089] The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0090] The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0091] Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the transporter peptides of the present invention as well as being encoded by the same genetic locus as the transporter peptide provided herein. The gene encoding the novel transporter protein of the present invention is located on a genome component that has been mapped to human chromosome 6 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0092] Allelic variants of a transporter peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the transporter peptide as well as being encoded by the same genetic locus as the transporter peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel transporter protein of the present invention is located on a genome component that has been mapped to human chromosome 6 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

[0093]FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 116 different nucleotide positions. Some of these SNPs, which are located in introns and 3′ of the ORF, may affect control/regulatory elements.

[0094] Paralogs of a transporter peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the transporter peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

[0095] Orthologs of a transporter peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the transporter peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a transporter peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

[0096] Non-naturally occurring variants of the transporter peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the transporter peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a transporter peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

[0097] Variant transporter peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind ligand, ability to transport ligand, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

[0098] Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

[0099] Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as transporter activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

[0100] The present invention further provides fragments of the transporter peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

[0101] As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a transporter peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the transporter peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the transporter peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

[0102] Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in transporter peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

[0103] Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0104] Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y Acad. Sci. 663:48-62 (1992)).

[0105] Accordingly, the transporter peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature transporter peptide is fused with another compound, such as a compound to increase the half-life of the transporter peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature transporter peptide, such as a leader or secretory sequence or a sequence for purification of the mature transporter peptide or a pro-protein sequence.

[0106] Protein/Peptide Uses

[0107] The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a transporter-effector protein interaction or transporter-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

[0108] Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0109] The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, transporters isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the transporter. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, and pheochromocytoma cells, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. A large percentage of pharmaceutical agents are being developed that modulate the activity of transporter proteins, particularly members of the chloride intracellular channel subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. Such uses can readily be determined using the information provided herein, that known in the art and routine experimentation.

[0110] The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to transporters that are related to members of the chloride intracellular channel subfamily. Such assays involve any of the known transporter functions or activities or properties useful for diagnosis and treatment of transporter-related conditions that are specific for the subfamily of transporters that the one of the present invention belongs to, particularly in cells and tissues that express the transporter. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, and pheochromocytoma cells, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems ((Hodgson, Bio/technology, Sep. 10, 1992, (9);973-80). Cell-based systems can be native, i.e., cells that normally express the transporter, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the transporter protein.

[0111] The polypeptides can be used to identify compounds that modulate transporter activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the transporter. Both the transporters of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the transporter. These compounds can be further screened against a functional transporter to determine the effect of the compound on the transporter activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the transporter to a desired degree.

[0112] Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the transporter protein and a molecule that normally interacts with the transporter protein, e.g. a substrate or a component of the signal pathway that the transporter protein normally interacts (for example, another transporter). Such assays typically include the steps of combining the transporter protein with a candidate compound under conditions that allow the transporter protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the transporter protein and the target, such as any of the associated effects of signal transduction such as changes in membrane potential, protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

[0113] Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

[0114] One candidate compound is a soluble fragment of the receptor that competes for ligand binding. Other candidate compounds include mutant transporters or appropriate fragments containing mutations that affect transporter function and thus compete for ligand. Accordingly, a fragment that competes for ligand, for example with a higher affinity, or a fragment that binds ligand but does not allow release, is encompassed by the invention.

[0115] The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) transporter activity. The assays typically involve an assay of events in the signal transduction pathway that indicate transporter activity. Thus, the transport of a ligand, change in cell membrane potential, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the transporter protein dependent signal cascade can be assayed.

[0116] Any of the biological or biochemical functions mediated by the transporter can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the transporter can be assayed. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, and pheochromocytoma cells, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

[0117] Binding and/or activating compounds can also be screened by using chimeric transporter proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a ligand-binding region can be used that interacts with a different ligand then that which is recognized by the native transporter. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the transporter is derived.

[0118] The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the transporter (e.g. binding partners and/or ligands). Thus, a compound is exposed to a transporter polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble transporter polypeptide is also added to the mixture. If the test compound interacts with the soluble transporter polypeptide, it decreases the amount of complex formed or activity from the transporter target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the transporter. Thus, the soluble polypeptide that competes with the target transporter region is designed to contain peptide sequences corresponding to the region of interest.

[0119] To perform cell free drug screening assays, it is sometimes desirable to immobilize either the transporter protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

[0120] Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of transporter-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a transporter-binding protein and a candidate compound are incubated in the transporter protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the transporter protein target molecule, or which are reactive with transporter protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

[0121] Agents that modulate one of the transporters of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

[0122] Modulators of transporter protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the transporter pathway, by treating cells or tissues that express the transporter. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. These methods of treatment include the steps of administering a modulator of transporter activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

[0123] In yet another aspect of the invention, the transporter proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the transporter and are involved in transporter activity. Such transporter-binding proteins are also likely to be involved in the propagation of signals by the transporter proteins or transporter targets as, for example, downstream elements of a transporter-mediated signaling pathway. Alternatively, such transporter-binding proteins are likely to be transporter inhibitors.

[0124] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a transporter protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a transporter-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the transporter protein.

[0125] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a transporter-modulating agent, an antisense transporter nucleic acid molecule, a transporter-specific antibody, or a transporter-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0126] The transporter proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. The method involves contacting a biological sample with a compound capable of interacting with the transporter protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0127] One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

[0128] The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered transporter activity in cell-based or cell-free assay, alteration in ligand or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0129] In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

[0130] The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the transporter protein in which one or more of the transporter functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other ligand-binding regions that are more or less active in ligand binding, and transporter activation. Accordingly, ligand dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

[0131] The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. Accordingly, methods for treatment include the use of the transporter protein or fragments.

[0132] Antibodies

[0133] The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

[0134] As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0135] Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0136] In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

[0137] Antibodies are preferably prepared from regions or discrete fragments of the transporter proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or transporter/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

[0138] An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

[0139] Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0140] Antibody Uses

[0141] The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, and pheochromocytoma cells, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

[0142] Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

[0143] The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

[0144] Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

[0145] The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

[0146] The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the transporter peptide to a binding partner such as a ligand or protein binding partner. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

[0147] The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nucleic acid arrays and similar methods have been developed for antibody arrays.

[0148] Nucleic Acid Molecules

[0149] The present invention further provides isolated nucleic acid molecules that encode a transporter peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the transporter peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

[0150] As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

[0151] Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

[0152] For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

[0153] Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIGS. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO: 2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

[0154] The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIGS. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO: 2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

[0155] The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIGS. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO: 2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprise several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

[0156] In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

[0157] The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

[0158] As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the transporter peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

[0159] Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

[0160] The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the transporter proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

[0161] The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3.

[0162] A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

[0163] A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

[0164] Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel transporter protein of the present invention is located on a genome component that has been mapped to human chromosome 6 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0165]FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 116 different nucleotide positions. Some of these SNPs, which are located in introns and 3′ of the ORF, may affect control/regulatory elements.

[0166] As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. Examples of moderate to low stringency hybridization conditions are well known in the art.

[0167] Nucleic Acid Molecule Uses

[0168] The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. As illustrated in FIG. 3, SNPs were identified at 116 different nucleotide positions.

[0169] The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

[0170] The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

[0171] The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

[0172] The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

[0173] The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel transporter protein of the present invention is located on a genome component that has been mapped to human chromosome 6 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0174] The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

[0175] The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

[0176] The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

[0177] The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

[0178] The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

[0179] The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, and pheochromocytoma cells, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

[0180] Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in transporter protein expression relative to normal results.

[0181] In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA include Southern hybridizations and in situ hybridization.

[0182] Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a transporter protein, such as by measuring a level of a transporter-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a transporter gene has been mutated. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, and pheochromocytoma cells, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

[0183] Nucleic acid expression assays are useful for drug screening to identify compounds that modulate transporter nucleic acid expression.

[0184] The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the transporter gene, particularly biological and pathological processes that are mediated by the transporter in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. The method typically includes assaying the ability of the compound to modulate the expression of the transporter nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired transporter nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the transporter nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

[0185] The assay for transporter nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the transporter protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

[0186] Thus, modulators of transporter gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of transporter mRNA in the presence of the candidate compound is compared to the level of expression of transporter mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

[0187] The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate transporter nucleic acid expression in cells and tissues that express the transporter. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, and pheochromocytoma cells, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

[0188] Alternatively, a modulator for transporter nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the transporter nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetal brain.

[0189] The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the transporter gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

[0190] The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in transporter nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in transporter genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the transporter gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the transporter gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a transporter protein.

[0191] Individuals carrying mutations in the transporter gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 116 different nucleotide positions. Some of these SNPs, which are located in introns and 3′ of the ORF, may affect control/regulatory elements. The gene encoding the novel transporter protein of the present invention is located on a genome component that has been mapped to human chromosome 6 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

[0192] Alternatively, mutations in a transporter gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

[0193] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

[0194] Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant transporter gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

[0195] Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

[0196] The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the transporter gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 116 different nucleotide positions. Some of these SNPs, which are located in introns and 3′ of the ORF, may affect control/regulatory elements.

[0197] Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

[0198] The nucleic acid molecules are thus useful as antisense constructs to control transporter gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of transporter protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into transporter protein.

[0199] Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of transporter nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired transporter nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the transporter protein, such as ligand binding.

[0200] The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in transporter gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired transporter protein to treat the individual.

[0201] The invention also encompasses kits for detecting the presence of a transporter nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that the transporter proteins of the present invention are expressed in humans in embryos, placenta, uterus and ovary tumors, eye (lens), testis, and pheochromocytoma cells, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting transporter nucleic acid in a biological sample; means for determining the amount of transporter nucleic acid in the sample; and means for comparing the amount of transporter nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect transporter protein mRNA or DNA.

[0202] Nucleic Acid Arrays

[0203] The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS: 1 and 3).

[0204] As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

[0205] The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides that cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

[0206] In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

[0207] In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

[0208] In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

[0209] Using such arrays, the present invention provides methods to identify the expression of the transporter proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the transporter gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the transporter protein of the present invention. SNPs were identified at 116 different nucleotide positions. Some of these SNPs, which are located in introns and 3′ of the ORF, may affect control/regulatory elements.

[0210] Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0211] The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

[0212] In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

[0213] Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

[0214] In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified transporter gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

[0215] Vectors/Host Cells

[0216] The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0217] A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

[0218] The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).

[0219] Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

[0220] The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

[0221] In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0222] In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0223] A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0224] The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

[0225] The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

[0226] The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

[0227] As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterotransporter. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

[0228] Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0229] The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

[0230] The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0231] In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

[0232] The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0233] The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

[0234] The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

[0235] The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0236] Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

[0237] In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

[0238] Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

[0239] While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

[0240] Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as transporters, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

[0241] Where the peptide is not secreted into the medium, which is typically the case with transporters, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

[0242] It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

[0243] Uses of Vectors and Host Cells

[0244] The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a transporter protein or peptide that can be further purified to produce desired amounts of transporter protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

[0245] Host cells are also useful for conducting cell-based assays involving the transporter protein or transporter protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native transporter protein is useful for assaying compounds that stimulate or inhibit transporter protein function.

[0246] Host cells are also useful for identifying transporter protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant transporter protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native transporter protein.

[0247] Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a transporter protein and identifying and evaluating modulators of transporter protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

[0248] A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the transporter protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

[0249] Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the transporter protein to particular cells.

[0250] Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

[0251] In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0252] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[0253] Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect ligand binding, transporter protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo transporter protein function, including ligand interaction, the effect of specific mutant transporter proteins on transporter protein function and ligand interaction, and the effect of chimeric transporter proteins. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more transporter protein functions.

[0254] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

1 5 1 2033 DNA Human 1 cgcggatcct gtgacacctc cgggcagccc ggcacttgtt gctcccacga cctgttgtca 60 ttcccttaac ccggctttcc ccgtggcccc ccgcctcctc ccggcttcgc tccttttcat 120 gtgagcatct gggacactga tctctcagac cccgctgctc gggctggaga atagatggtt 180 ttgtgaaaaa ttaaacaccg ccctgaagag gagccccgct gggcagcggc aggagcgcag 240 agtgctggcc caggtgctgc agaggtggcg cctccccggc ccgggacggt agccccgggc 300 gccaacggca tgacagactc ggcgacagct aacggggacg acagggaccc cgagatcgag 360 ctctttgtga aggctggaat cgatggagaa agcatcggca actgtccttt ctctcagcgc 420 ctcttcatga tcctctggct gaaaggagtc gtgttcaatg tcaccactgt ggatctgaaa 480 agaaagccag ctgacctgca caacctagcc cccggcacgc acccgccctt cctgaccttc 540 aacggggacg tgaagacaga cgtcaataag atcgaggagt tcctggagga gaccttgacc 600 cctgaaaagt accccaaact ggctgcaaaa caccgggaat ccaacacagc gggcatcgac 660 atcttttcca agttttctgc ctacatcaaa aataccaagc agcagaacaa tgctgctctt 720 gaaagaggcc taaccaaggc tctaaagaaa ttggatgact acctgaacac ccctctacca 780 gaggagattg acgccaacac ttgtggggaa gacaaggggt cccggcgcaa gttcctggat 840 ggggatgagc tgaccctggc tgactgcaat ctgttgccca agctccatgt ggtcaaggta 900 agagagctct acccacaggg gcctgcaaga tccagctcca tcttaggccc aggtcacctg 960 tgtggatgag tcaaggacag taccacctgt tggtcaagaa cctggaccct gaagtcaggt 1020 aataaggacc caaggtcacc ctctgctgct tgttggctgt gtggcctcct tgagcttcag 1080 ttttcattta taaaataggc atatattgct tacttcaagt gttggtggaa gagtaaatac 1140 agcgtgaaag tgcttggcat attgtggggg cttaatatgt gtaatagtcg caattatcgt 1200 tgttgtatac agtcatatca ctccaaaggc ctcttcctca taggatttcc ctggctacac 1260 ccctacagct ctattaaatg tgccctcata tgcatttttt ctttgtgcac agacccacct 1320 tctcacttcc tccagcaact tcctaaggtg agcccacatt attttcctca tctatcaaat 1380 gaagaggtgg aggttgcaag aagtgatgtc actttcttgc tatcattgca cttactaacc 1440 atttgcagca tgtagtgtca tcctctccta tataacaaac cctgggaatc tgagagttgg 1500 aaaggacatt tagaggtcat ccaaaacaat ctcccacttc aacctggacc actttctgct 1560 gtttctgtga caggcttctg tgaagctgtg actgcagcct ctgagaacga ggagccctct 1620 gcttaatgag ccagccctac catgttagat agctctgatt attaaatcat tgttctttac 1680 aatgagccca agcatgcctc cctgctatcc attttttctc tagagtaaca gagaacagct 1740 ttgcttgcct tcacctcatt tgaagacagt agttgtatcc ccctaagctc tgtcaatgag 1800 cacttcttcc cccattcttt cctgaccctc gtcagcctag tatcagatag ccatactgtg 1860 ctctatttta cgcatgctta tatcttactg tctcagccag acagcaaact ctgtgaggaa 1920 aggacctttt taaagtgtga tggtgggcac acagtggcca ttcaataaat actcattgat 1980 tgatcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 2033 2 219 PRT Human 2 Met Thr Asp Ser Ala Thr Ala Asn Gly Asp Asp Arg Asp Pro Glu Ile 1 5 10 15 Glu Leu Phe Val Lys Ala Gly Ile Asp Gly Glu Ser Ile Gly Asn Cys 20 25 30 Pro Phe Ser Gln Arg Leu Phe Met Ile Leu Trp Leu Lys Gly Val Val 35 40 45 Phe Asn Val Thr Thr Val Asp Leu Lys Arg Lys Pro Ala Asp Leu His 50 55 60 Asn Leu Ala Pro Gly Thr His Pro Pro Phe Leu Thr Phe Asn Gly Asp 65 70 75 80 Val Lys Thr Asp Val Asn Lys Ile Glu Glu Phe Leu Glu Glu Thr Leu 85 90 95 Thr Pro Glu Lys Tyr Pro Lys Leu Ala Ala Lys His Arg Glu Ser Asn 100 105 110 Thr Ala Gly Ile Asp Ile Phe Ser Lys Phe Ser Ala Tyr Ile Lys Asn 115 120 125 Thr Lys Gln Gln Asn Asn Ala Ala Leu Glu Arg Gly Leu Thr Lys Ala 130 135 140 Leu Lys Lys Leu Asp Asp Tyr Leu Asn Thr Pro Leu Pro Glu Glu Ile 145 150 155 160 Asp Ala Asn Thr Cys Gly Glu Asp Lys Gly Ser Arg Arg Lys Phe Leu 165 170 175 Asp Gly Asp Glu Leu Thr Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu 180 185 190 His Val Val Lys Val Arg Glu Leu Tyr Pro Gln Gly Pro Ala Arg Ser 195 200 205 Ser Ser Ile Leu Gly Pro Gly His Leu Cys Gly 210 215 3 106323 DNA Human misc_feature (1)...(106323) n = A,T,C or G 3 gtagtgagga catctttccc ttttctttgc ctatcagatg cctgaccttt caggaaggtt 60 cagctaaagt gccttttgct ccatgaagcc ttttcctctc ctcaccccaa ccatggtaaa 120 tagtatcttt acgtgacagc ttgtatagct ccttcatctt cccaattcat gtggcagcct 180 tagttttcat ttcatgtaac tattattctg aaagggattt tgatctctgt taagaccgtt 240 aaaaccatgc ctttgtttcc tcaaagtgtg cacttccata gtgtgcacta agaaaagcag 300 tttgtagagc agttgagagg acaggctcaa gtctgtctgg gtttaaatcc tgacttcacc 360 atttcctagc tgaatgacta gggataagtt acttaaatac ttcatgtctc agtttcccca 420 cttgtaaaat tgggatcatt atactatttg taaatattat acaagttcac acatgtaaag 480 tgctcaacat ttttgcctag cacataataa gtattattac ttattttttt attaagcgat 540 aaatttttta ataaagctat taaacaaata catgttttta gtaaggaagg acatagcaca 600 aacaggatta attaaagaga aggcaaagaa gacaaagaga attccaccag attctggtgc 660 aaaacattca ataattattt tttaaagaac tgaagggtgg gaatgtctgt tggtgcaacc 720 actttagaaa acctatggca gtgtttacac aaggagagca ttcttataca ctacaactca 780 gcaagtccac tcctaagtat acatccaaga gaaaagtgtc tgtgaatcca accaaaagat 840 gtttgataga gagaatgttc gtgacagagt agtcatgata gtcacctgca caaatgtcca 900 ttgacagaag aatgaataag taagtgtgtc ggtgaaaatg gatgatacac aactacacat 960 agcacaatgg atgaatctca caaacatagt aatgagcgaa agaagccaga caccaacatt 1020 ccaggcaaaa agatcctatg ctgctacaag ccagaataat ggttaccctg ggacagtgga 1080 gacgggagaa aagatgtgga gaggagagag caggaggagc ttctagggct gtggctaaca 1140 ttttattctt catctggcgc tcgttacatg ggtgggttgg attgtgaaaa ttcattgagg 1200 catacaccta agatatgtgc atcttttctg tgtttgcatc ctacttcact aaaacaacca 1260 gaagagaacg ggaatgaagt aagatcagaa aataaaggaa gaaatagaaa gaaaatgaaa 1320 gaagggagaa gaaaaataaa caaacccaga aggctgaaag gcatggaagc cgcacctgac 1380 cgggttgact cacccctctg ccccaggagc cacccctgcc ctgccctgcc cgtccccagc 1440 accctactct ccttttgctt agcccaagga tggcaaatgc cccttttggt gggggttctg 1500 agcatcacta agacaggaag ggctttccaa gtgggtaagg ctggtagtgg agtaactcgg 1560 aggagctgac gctttattaa agaaacagaa gacttgagag ccatctcctc tcttccttcc 1620 ccttcccctt tccccttcct cttccccttc ccctttcccc ttcctcttcc ccttcccctt 1680 tccccttcct cttccccttc ccctttcccc ttcctcttcc ccttcccctt tccccttcct 1740 cttccccttc cccttctcct cgactctttc tgccttcgcc tcattatccg cccccaggac 1800 atcctcttgg ccttggtggc tttgccagga ccagctctgt ctccgcctcc tgccctgcgg 1860 ggtacttggg gcggggggcc gtggcccggc ccaggcccga gctgccgggg tgtccccgcc 1920 gcgtccccgc ccggctgacc ccgccctgca gccgccggct attttgggcg cgttggcggc 1980 ggcgggatcg ctgacagtcg cggatcctgt gacacctccg ggcagcccgg cacttgttgc 2040 tcccacgacc tgttgtcatt cccttaaccc ggctttcccc gtggcccccc gcctcctacc 2100 cggcttcgct ccttttcatg tgagcatgct gggacactga tctctcagac cccgctgctc 2160 gggctggaga atagatggtt ttgtgaaaaa ttaaacaccg ccctgaagag gagccccgct 2220 gggcagcggc aggtagcgca gagtgctggc ccaggtgctg cagaggtggc gcctccccgg 2280 cccgggacgg tagccccggg cgccaacggc atgacagact cggcgacagc taacggggac 2340 gacagggacc ccgagatcga gctctttgtg aaggtaggtc ggggtccagt ctcccgccgc 2400 acctgccgcg cctcccgccg ggctcaccca ccagggctcg gggtgcgctg gactccggac 2460 cctcccagga gggcggcgcg gagccctgtg ctcccccgtg gcctttccgc aggccagcgc 2520 cccgctgcga ggctcctggg cggaatggag gaaggcaggt tccggcaggt ggccgtcttc 2580 tcggagagtt gatccgcagt ttgtccaccc ccgtccccgc ctccgctgtc accgctgcga 2640 gtccgagtcc agagcccagc ccggacgcga ttcaggctgg tcctgaaaga cgtctccagg 2700 tgtcgggatg cggcagaaac ctggctggac ccgtgaacct ttcctttaaa acgggaggag 2760 ggggaggaat gggagagaaa cgttattgac acaatgatct ttggcaaagc gaaaatgaca 2820 tttcttaaaa ttctctttct ctctttcgct cagagatcct aaaataaaag ccaaggagac 2880 aaaagtgttt tcttgcttgg tgacttgcaa agcagaaaac aagcactcca ttctccgacg 2940 ttattatttt tgtcataatc atgatttttc aaggcagtat caaggaaatg ttaggcgttt 3000 ggtagtaggg agagcctccc gttcaacctg atgcagacag ggatgaagga gcacagtgca 3060 aattaaatga agtcaccttg ctccttggac agaaactgcc taatgtcaga gctgctgtac 3120 tttatttgca acctgtagga ggaaggatgg aggcgaggct gcggcggcgt ttcccggtcc 3180 gccggtgtaa ggagaaagaa acatttgatc agcccttggt gccgacgctg cagaagtagg 3240 gaaacctgag gctgcgggag agtagcctcc agggctgagc agaaacagtc gccaggactt 3300 gggggagggg aagtcgggga ctgcctgggc gccaagacct ttcacaaagg gggaaaactc 3360 tctcagtgca aagtttccct agaaatatgc agatcgctga gaagggaaga gtcactggtg 3420 cctgtcaggg gcctcctggc agaatttaaa aggcataaat attccctgga attgggaaaa 3480 tgttattttg cccaaaagat ccttctctac aagctaatta aatagatgtt tgcaaaaagt 3540 atgagatcta cataaacctt gataacaatc tgtggtcttt ggtactttct gactcttaaa 3600 atgtgactta ctttacactt ggtacgtaat ctgtctccgt tgtcccagcc taccacaaag 3660 gtttgttaaa tttcttctgc tgggttttac attctttgtg tttatttttc aagcttacac 3720 ctgacattta gtgacacttg ctttcttcag cctgttctct tcccactgtg gcccctgtgt 3780 gctcctttgg tttctgggac tccccccacc cccaccccag gctggatttc caggtccttt 3840 ggcagaccca ggtaataaag gaggtcattt ctcgtctgat gctgggtagt gttggacctt 3900 agggcagcaa gggggtgttc tgaaccccag catccaggcc tgggccctag gggtgaatag 3960 ggagagcttt aacaaagcga tgagtcttgc tgctctcctt gaagaaagcg attctaggac 4020 ttactcagat tccttgagag tggtagtttc aaggtgtcaa gtttctgaaa gatatttgaa 4080 acacaaatag gttctttaga accaaaggag ccctttgccc cactcctagg tgtggagacc 4140 gattctacct gcacctttgg aattttcttg ttggcagtgt tggagagtct ctccatttat 4200 ataaaaacct tactgcacaa tttgcagcgt tccccaaata attctaaaat gactgtgttg 4260 tcatttgttt tttccctaaa ggaaatcaat gagagccagg caggccttca tggctagctc 4320 aaaaaccaga ttgatgggga gatcaaaaca tcaggtatga aagttgtttt gactcctccc 4380 tcaaccccat ctttttgagc ttcaggttag cagttctcat ttacaagtaa atgggacaag 4440 taaaggctag aaatattgtg aaataaagat gctgcccagg ccttacttga actgtgtcat 4500 ttgagggaag caatatcctt gttgacagaa tcacactttt ccaaagtttg gagtttctct 4560 gaggtacatg attccccata aaaagaaagg ccttcttgtt gatggtgtca ggtaactgaa 4620 gctctttctg cagggcaggg tgggttggtt tctatgaaga cttccaagaa ggatgggtcc 4680 cacccagtta tagtcatgac ttggtcattt tcaacacccc cttggaagga cctcccaacc 4740 tcttgtttcc actgggtgta ccactctaaa tcacagtcac ctaatgagtt tcacaatgac 4800 atgtaattgt aattgtctcc cacgtcaatt gatagtcatt atctgttact actactgctg 4860 ataatgataa tgataagaag aaggaaagat tgcagcatat taggacataa taagtatgtt 4920 caaatacctt atctcatttg atcatcagca actttataag gcttgattca cttatgtgaa 4980 agataaagaa aacaaataaa ctggattcac aggtacctta tgagactcgc tgtcagagca 5040 cttgatgagc tgcagtttgc tcttcttgta acagcccagg gaactaagaa tgataattcc 5100 tcctgttctg ttgaacaata catgtgctca cagaaagtag gtaaaatgcc caaaggtaga 5160 tggcagatag cattcagatc cagtcctcaa atctagcagt ttagactttg aatttcttgc 5220 tcttttccaa atacctatat gtcaggcctg ataccttatt ttgccaagtt aggctttcaa 5280 gtgccatcat ggtgacattt atttatgtta ttaatccatt ctaatgtgtt catagaatgc 5340 ttccaaggag actaacccat ttctgctatg tactccgagt ctttcccttt cggggaacat 5400 tctgacagtg acttttaaga gattatttcc ccacattttt agtttctaca ataagaaatg 5460 ttagatcatg tcaactctga ttctaatgtc ccataatacc tgtggatggc gcagggattt 5520 ggagatgcag tacctgagtt caagtcttgc ctctgcctct aattagctaa atgatcctga 5580 acaaattatt taactttcag gagcctcact ggccctctta aaacaagagc atggttataa 5640 ataaggagac catataattt attgtccaaa tctagacact tttgagagtg aaagggaatg 5700 atatcaataa ttaggctgga accacaggta taacccagga tagtttggga gcaaataagg 5760 atgtgtggtc attctggtta tgaaaatact gtcattgtag atttgctgtg aggattccat 5820 gggagaacat aggtgaaaac actttgtaaa ttgaaaattg ctgtaaacac actgacttga 5880 atattatgag tcccatggca gactgaacct ttctggacct gcacgtccct gatttgcctt 5940 ggggaacaaa ggcctggctc cttcctcact tgcagggata ttttgagagt aaatgagata 6000 atatgtagct tggactttga tcctaagagg aggacaacag gctagaggta atggcagtga 6060 gtgtggttta ttgaaagaaa tagagcaagg aagtaaaagc aaaacagaaa gcaatttgaa 6120 aagacaaaca caggatgtgc tgtgtaggtt agatcaaggc attccaccca tttgcattgc 6180 ttcctccact cccagtctca cccctggacc ccagaagtga ctaatgatac agacaagctc 6240 gccgcaagtg tctcttcagc caggaactga ggtttcaaca gaaaccagtg gttctcagcc 6300 ttcactacct attcaatcca ttgagagaga ttttaaaact tctgatggca gtttgctccc 6360 taagccgatt acatcagtgt gtctgtgggt gggaccctga aacaatattt tcatatccac 6420 tctcaggtgg ctccagcgtg cagccctggt ggagagccac tgagtcataa tcagaggcag 6480 gtccaatagc aggcagcttc tgccaccgct gacataggtg gccttgctaa tgctgattaa 6540 atgtccagaa cttctaacag cctcagggca tcacggtcag aagtgttgtt gatagaaagc 6600 ttgcatccaa tcactgccca gttaacagat gctgacgaac tattaagccc tatccacagg 6660 actccagaac aatcgtgtat agggttggtc tagtcaaaga atcatcaatt cagcatgggg 6720 gaagtaaaac agagttctgc taattgtctg atggaaacga ctagtctttc atttattgag 6780 tggcccaaaa gtttctgagt ggtgcagggt gcaatacaag ttgtggtgac tcgtggtgtg 6840 gtcatctgac ttccagaaca gataggatca gatcttgtgg tgtcgcctct atgtccctga 6900 gctactgtga ctgcctggtc accttgggcc gcttgcctaa gcatgagctc actaggtttt 6960 taaagttccc ttcatcatat taagctactg tgtttttctt tcccactatc ttcaggtttt 7020 gatctgtagt caataacgca atacacaaat ttgcctgccc tgctatggca gtgagtgctt 7080 cttctcctgg taacacatcc ctggagagct agcgtcaaga gttgtatgat gcatatttga 7140 agtgccatgg cagggtggtg ggaatgagga ctgggtctgc agaagctgac gtccactgcc 7200 agtgttgaga gctcctgcat ccctgatgac ttgtgttgtc tctttgggca tttattggcc 7260 ccctccatgc aagacaaagc gtctgtcaat ttagacagtc agttaggtca aaaacttctc 7320 attcttggtt ccctttgagt tgtcagctgc tggccccatg ttagtggtgg caagagatgt 7380 ggtggaagag tgaaactttg gtgtcaggct cgggttcacc agtgtgttgg ccaggatggg 7440 ctgcttgacc tctctggacc acagattttt tttttgtttt aaccagtgca gtgctaatag 7500 cagcagcaca tctttcatgg agttggtgtc agcatttcaa gctgagtatg gatctggcct 7560 attagtaagt ctttctctgt aggctccatg tagcccaata agtaaaagta aagcccttgg 7620 gcttccctcc cttgatgtgt acaagaagtc agaggctcag tcctaccagc tgaatctcac 7680 atctcagaac caagatactt cttgaaagaa aatgtaagtt tccaaagcag gtcgtcattc 7740 agggaactgg aagagaaatg ttcccgattg ttggagctga cccctgagaa cacaagcagt 7800 gagtctcatt gctgcagacc aatggcaact gaagtacagt gcaaccacca ctggatctga 7860 ctgcctctga ctggactgct gctccataag ctggagccca ctagagccca tgtctggagt 7920 aatgtggttg gactgactac agggtaactg cagatccctg acccactggg ttcttcaaac 7980 tcaggggaag ttgattcctc ctgtcagttg ctccctcatc caagaaagct cctacataac 8040 tgcttcccca aggctcctgg tagccaggcc agggtaaagg atcctcttta ttttcaagag 8100 gatccctggt tctctttgca gattttacta ccccttcctc aacaatcttc cagctatgca 8160 tagccatagg tgctctcatc aaatatactc acacatgcac acgtattatc taccaggatt 8220 atggggaaaa tgtcgtaggc tctcactctc tggtatctat taaaaaacta ttacccagaa 8280 cacatcctga gttgtatctg ttgtcaagac tggaggaact agagatagta aagatgcaaa 8340 gtggttaagc atgagtttcc taagatgtag ttctgtaaag gactctctct acccagggtg 8400 tcttaaatct aacactgctt gtgttctgag gggtcagctc ccacagtcag ggacatttct 8460 cttccagttc gctaaatgac aacctgcttt ggaaacgtgt cttttctttc aagaaagaag 8520 tatctaagtt ctgagatgtg aaattcttta aggacaaata agataaatag cctactgtct 8580 gaactgaaac gaattcttcc ctgtaaaaag aaatggagtt gttaacttct agaagttctt 8640 ttagctctgc tggcatggga agagtcttta agggggattc aaagactctt cccatgccag 8700 cagcactgga ggtaaaccat gggtaggctg gtggttctca gcctttgggg catgaaagag 8760 tcactgcact tgccttcttc acttgtacct gcccaggtcc tgtctataga gtttcagatt 8820 taggagggtg gggtgaggcc caggcatctg tatgcttaag aagctaccca ctggtgattc 8880 tgaagcccat caaagactga gaaccactgg tgcaaaccat gggtaaatag ggctgaacta 8940 ctcagtggag catcaaaagg acaaaatatg gtccagtcgc tacttcaaac aagcatttca 9000 caggatggag gtagccaagg caatatgctg gttcacatga atcactccag attgacctca 9060 aataaaccag aaaagggagg aaagaaatgc ttttgacctt tcttttgtaa tttttctttt 9120 cttcctttgt atatttttta ttatatagga gtgacacata attttaaatg gcaatgcatg 9180 aaggagatag taagataaag aatgtaggga gttggggata aaatataatc aatcaatcaa 9240 tatatgaaaa tgcttacaga gcacctactc tgtgccaggc tctgtgaaag ctgaaggggg 9300 ctgggggaga tcagagaaga agacaggctc cagtagcttg tgagagtgcc actgaggagt 9360 gcaatgcaaa caagcatcag tggcacatga gtgacaggaa tgcaatgagt catgcatatg 9420 atgggaccag tgtggcccaa atggtgtctt caggcccagc tttgggtagg gtgggacctg 9480 agttgagtct tgaagaatga tcaggattca gaggtggcca aaagtgagca accatgtcag 9540 gtccagcaaa ggtgtggaga tggggatgct cagggcttat gagtacacgg ggttgtctga 9600 agcaagtggt atatgaaagg agcaattgga aataggacag caaaaaagcc aacaacagtg 9660 gcaggagggg gccttgaatg ggaggtaagt ggtatctgct cccttgtaag tagtggggag 9720 taactggaag tgtctacaca caataggaga ttgacttggg ttatgtttag aaagattgat 9780 agctggactt cgtgctttag tgggaaaatc aatgaactca aagtatgatg gataccctaa 9840 aagtcctgac ttgatcactg cacaatctat gcatgtaaca aaattgctca tgtaccccat 9900 aaatttgtgc aaatgaaaat cgggaaaaaa aaaaaaagaa aaagaaaaaa aacaggaaaa 9960 aacaaacaaa caaaagaaag aagatctgga ttccaggtct gccacgcact ggttgcgtga 10020 cttgctttaa gggtaagttt tttttttcta taaaatgtga tcaacattca taaaaccata 10080 gaatttatta tttggaaaag cttccagaag tcatccagtc tgaccttctc tgatgagata 10140 agttcccaat ctaacacttt gggatatacg ctatgtgggt ttaaggaaga gtagtgagat 10200 aaaatgtgag ctgctgctgt ttttgccatt gctattacgg ctgcattcac tgtgaccatg 10260 ttggaataag gagagccctg aggcagaagt cagtgaggag gctatcccag tggtcccatc 10320 aggaggccaa gtgagtccaa acaagggcca aaagacagag tgtgttgtgg aatacagaag 10380 tgaatacaaa attaggtgca tgccttggaa gaagcctgtg caaatagtgt atattagggg 10440 caccaacacc gaagactcag cagtttcctg gtacattcac ccctggaaag gttagtgtcc 10500 acatctagaa tggtgatatc tttgatgggc ttgaccatta ctgtctaaga tattgtaaat 10560 tgaagagtac agacaaacta attgagcttt aagtttcctt ttaactctga agaaagccta 10620 tgtatcctgt gaggaaggga gttgctgttc attaaaatcg tttgatttgg aatggtgtaa 10680 tctgaagtgc tggcaggcca gtcctgtgca gatgttcctt agaatgctga aggatcagag 10740 ctgaggatca caggggctca gggctaggga ttttatctga acttctgctc agagctacag 10800 gtgacggagg aggttcccca gaggagggag catggagaag aagtacagaa ataaggactt 10860 gctttaaaaa atgtcttttg gggctgggca tggtggctca tgactgtaat ctcagcactt 10920 tgggaggccg aggcaggggg atcatgtcag gggatcgaga ccatcctggc caacatggtg 10980 aaacgccgtc tctagcaaaa tacaaaaaaa ttagccgggt gtggtggcac ccacctatag 11040 ccccagctac ttaggagact gaggcagggg aatcgcttga acccgggagg tggatgttgc 11100 agagggctga gtttgcacca ctgtactcca gcctggtgac agagcaagac tgtctcaaaa 11160 aaaaaaaaaa ggaaaaaaaa gaaatatcat ttgagaggga ggaatgaggc acgtgaggag 11220 ttagccaagg tgatcaagga ataagaggta ggaaataaat gcaatgtcct aggcaaagga 11280 aatcatgtga acaatggctc agaagcaaga caagcccgga tgtgtcccag aaaactcaga 11340 gctctgcttg gctgtgctga agagggtgtg aatggatatt gggaaggcag ataaaaatgt 11400 aagttagggg ccaggcgcag tggctcacat cagtaatccc agcactttgg gaggctgagg 11460 tgggcagatc atgaggtcaa gagttcgaga ccagcctggc caatatggtg aaaccccgtc 11520 tctactaaag atacaaaaaa ttagctggac atggtggcgc atgcctgtaa tcccagctac 11580 tcaggaagtt gaggcaggag aattgcttga accaaggagg cggaggctgc agtgggccaa 11640 gattgcgcca ttgcactcca ggctgggcga tagggcaaga ttccatctaa atatatatat 11700 atatatatat atatatatat atatgtattt atacatgtgt atatatatat atatatatgt 11760 atttatacat gtgtatatat atatttatac atgtatatat atatttatac atgtatatat 11820 atttatacat gtgtgtatat atatatatat aaatatatat atatatatat acacacatag 11880 gctctgtcct aatctctttg agctgctatg acaaaatgcc atgaactagg tagcttataa 11940 acaacagaaa tttatttctc acagttctgg aggctgggga gtctatcagt gcaccaatag 12000 atttggtgtc tggtgagggc cacaatgtgg ttcatagctg tcaccttctc actgggttct 12060 cacattgtgg aggaggagaa ctctattctc ttcagcccct catgagaaca ctaatcccat 12120 ttgtgagggc tccaccctca tgacctaatc acctcccaaa gcctccacct gctaatgcca 12180 acacctcaga gatgaaattt tagcatagga attctggggg gacacaaaca ttcagatcta 12240 gcagatcaga tcatgaagca tctctgatgg caagctgggg gatccagact tttgtttata 12300 aagtggaaga ctgctgtgcc cctacctgcc tgtggagcaa ttgtagttta aaagagggaa 12360 gtgcttaatt atctaaacat ggtgcatttg gccctgcatc tgaggcaggg tgttttggcg 12420 tgggaggcct ttgcagcacg tctctggtta cccacaggct ttggtgtgta gaagttttgt 12480 gccagttacc ccatatccta tatcaacctc tatggacttc agggtgatgg tttcttttcc 12540 acttttgttt aacgttattc tatggtaaga gggaaaagaa acaagtgtcc gactggagaa 12600 ataacacaaa aggggaagga ggggaggatg tgcagtgaat ggagtccaac tgggaaccca 12660 ttacctagaa tagagacgaa gtaacaccat gtcccagtta ttctcttaaa catccaattt 12720 gggagattgt catggcaaca gatagaggtt tcttgatcca gcccaaggct cctctcctgt 12780 caatgtccac gtctgcttcc taatgctgcc agtgtgcatg ggccccgtca acaccaagga 12840 tgaagacgag gggccgtctc acctgccttt tattcattta cacatttgct ctctgtggca 12900 ttttatagtt atagcataaa atctagagac agcagtgtct ttagaatttg cccctgaaac 12960 caaaggagcc atatgtcctt ggccctttgt cacaaatgac tcaggtttta cttgtggaat 13020 tgaaggttct gggcagggct tccccacagg ctctgggaga ggggagctgt gtagggaaag 13080 ttatgtggcc tttcctcatt caggtacttt gtgaatataa cagggatctg atctgtgtgg 13140 caggattatt attatgaagt caaataagat aagaaacatg aacccctttt gcaaactaca 13200 gtcttcactg ttcagatgtc aggcactgcc ttggtcggta taggtcaact gctgaaacaa 13260 caacctacca actttttcac acagtagctc aatgcagtgt ttagtgagtg gtctccccca 13320 cttggactcc ctccaccctg tagttctgcc accctgcggg acggccaact cctttgctgg 13380 gctctctaca gtcagccagc acagggaaga ttgaagttgg aggacatcat ggtaggtttg 13440 catggtcgaa tcacttccac ccactttcca tagtccagag ctcaatcctg tggctgttcc 13500 tctgtgcaag ggaagctggg aagtgtcatc taactgtgct gcaaggacag agaggaagca 13560 ggtttggagc tagtctctac cgtagaaact aagttatgta ttcttgtttc ctactacaaa 13620 ctggcatccc acctgcagcc tctctcacct cctgttgacc atgcttaata ctgtccactt 13680 aatcttccta aagaagaacc ttctcctgta tgacatcgat tccccatctg ctagatcaac 13740 ttcaactgct tccacctgat actgaaacta ttcttgcatt ccttttccca gcgttatttc 13800 ccatattctg ttaaaatggt tccttgtgtg tacatacagt tttttgaagc atttttgggt 13860 ctcttatcct ttgaggaagg aagatagtta atcctctcat ataccaggtg atttgtcagg 13920 ggctgcatga tgggttgctc tatacaaact tagaaacctg gagatccgga ctccagacct 13980 gaccactctc caccagaaca gggtttccca gccttgcaca ggcatgagaa ccacctgggg 14040 cattttccaa actccagccc aaatgtgtga atcagagtct tcagggaagg aggtcccaga 14100 tacctgtaat ttaacaagtg ctgcaggtga ttcttgtgcc cagtctggtt aagggaacac 14160 tgcccttaac ctaaccaggc cccatgggag tatccaaaac ccacaggggt gagtctaaca 14220 gtggaaattt aaagccctgt gacattcaaa tgtaggagag atttggtagg cgagtggtct 14280 tctactgctc tcaggtcacc ccaaggtata taacatctca gtcattttat atagtgacct 14340 ttagacttag gatctgctcc atggcccgtg gaattataaa aacagttgca gccttgttta 14400 ttgttttgtt gttggtgttg ttgttgtttt tgcaaaggta tttatcccca tcctcaaagt 14460 ataaggttct tgaagaggaa aatgttggga aagagatagc ggggatggaa gagggaaaag 14520 ggtaggtgag acagaggatc actgaagtgg cttatgatac agaaaaacca gtcttcacac 14580 ctcagtcctc tttggtccca tgagtggtgt ctttgcattt tcaaaatcag agaatcagtt 14640 tggaaattga ataatagcaa cagtggctaa ctgctattct gaacacctta gtttcgctga 14700 ttccttgaat tccatggagt aggtcttggt actattccca atttacagat gaggaaactg 14760 aaggtcagac tgagtcccat ctggataaat gcacaaagct aggacattgt agagcctggc 14820 ttcaaacttc atcccctgac atcagaattt gtgctctctc ctgcctcatt ttacccagcc 14880 ccttgtagct taatgaggag aaattgctgc gctaaatcag aaggcatttt cccaatggcg 14940 agcaagaggg agatgaccaa ggttttgcct ttccttaatt ctccataggt ggcaatgaat 15000 agtccaaggt caaggtcaga tcccaacact tagacaagaa agcagggcaa ttaccagatt 15060 gggtgagtta gagcagccta aagacgcacc tgttctcaag tctcctaccc acttctctgc 15120 catagtgaca tgaactccca ttctttgatc aacccctttg gcttaaggaa tgtttcttag 15180 acgtcctttt aaaaatatac atatgctact tgtaatgggc tacctatgga tgcagtagcc 15240 ccagctgaca cattatctgg gattacttta tcaggtcagt aggcagaggg aagaggacca 15300 gaaggaagtg ggtaagagag accaggagta ttgcatgcca ctgggaaaaa aaagtgtacc 15360 tgttgtgcat tgtgggtgtg gccaccaaga gcagattttg tcccctggcc agtttgccca 15420 agatgtggca ttggcccaag ttcctcagtc aggattctag agtaagctct gaaaatcatc 15480 tttgcaagga tgaagccact gacttgagag aaatcttaca tctattccag tcggctcttc 15540 ttccattcta agacctagag tcatttacag atatgtatgg cagagtccaa caacaacaac 15600 aaaaaaaaat tacagactat tcactaggca ctataaatgt gagatataac aataaatcta 15660 aagaaattta caatatcgtc catggaattt gatggattcc atcgagtcct cgcaggattc 15720 tagcagcttc attcctcctt gaggcccaga caagttaggc tgttataatg ccacatagaa 15780 gactgaaagc agaggtgaga ataaagctgc tgctgattga gtttactccc tggtgtgggg 15840 ggtgggggtg gtccagcctc actgctagga ctttctgagt ctttctgaga cacttgccat 15900 ggtcaagggt agcaggatca gggaaggcat tataataaat ataatttgca gagcatctct 15960 ctcctatgca ccagatattg tggtgacact ctgtttaatc cagtatccct actcctttag 16020 atatattgtg attgttttac atgcgaaatc tggcttcaga aaggttaggt gttttgctca 16080 aggtcccatg gaaagtggca gagttggggt ttctgactaa ctccaaaacc ctttaatgtg 16140 tgttcataat taaaaacaat aaaaaatgaa gtcaaggtac ttgaaatgca cttttccatg 16200 tggcagctgt ttactgaagc ctaccaggtg ccaggccctg ggtcttgtgg gggaagccag 16260 ctgggacctg gcagaacttg cagcctgggg gtctgggaga gtggcagcca ctgggcattt 16320 ggaaggactg tggcctgaag gcaagaatga ggctatggga gctccaagcc aagggacctg 16380 gtatcaggaa ggcactaagc agcgttttgc agcacagcca gggaagaagt tggggttcag 16440 ttcaagtgtg caacatgagg ggagaggccc agctaagctc aggcagagaa gggggagaaa 16500 gccatccacc tcttcccctc tctcccccgt agacctgttg ctagggcaga ttccagagca 16560 ggtgataggg ggacaccagg ctctccaccc agtcaagacc ccaggacctt ctcttggtac 16620 ccacctaccc caggagagta aggacactgc tggagaaaaa ccatggacta tctcttctgg 16680 ttcctctccc agttaaggtc accctaaagg atgataagag gcttatctag gcctaacact 16740 cctcagaaag cattttccat ctgtatgcca agaattgctc taactagggc gaggcaactt 16800 tcattccaga gtgggagaaa aatgcctctc aaagggaatg ccttgttggt gaacactgta 16860 gagtgaagga atacccagac ttcatttcaa agagtggtta tcagatgcac ctagttgaca 16920 agaggtttgt gacatggggg atggtcaatg aagagctgga aaaagaggct ctgtgatatg 16980 gtttggctct gcatccccac ccaaatctca ccttgaattg taataatccc catgtgtcaa 17040 gggagagacc cgatgggagg taattgaatc ttgggggttg tttcccccat gctgttctcg 17100 tgatagtgag tgagtctacg agtctgatgg tttcataagt gtctggcatt tcccctgctg 17160 gcagtcattc tctctcctgc tgccctgtga agaggtgcat tctaccatga ttgtaagttt 17220 cctgaggtct ccccagccct gcggaaccgt gagtcaatta aacctctttt ctttataaat 17280 tacccagtct tgggtatttc ttcatagcag catgaaaaca aaccaataca ccagggatga 17340 aagacaaaga ccatccatgc atggctctgc tccttcggtt tggtgctgac ttccatcacc 17400 tgggttcaga tgaacaggtg gtaacaacta aaaatggtga ccctgaagtc acaaatcaga 17460 tggatactcc cctcacggtg ggtgtctcct tcaaaggagc atgtgcacca tcatcttatt 17520 attacacaga catacacagt atgtgtgtgt tttacatata tacagttaac ctccaaacaa 17580 cgtgggggtt aggggcactg acccctccat acaagttgaa aatccatgtg taacttttga 17640 cttcctaaaa tcttaactac taatagccta ctactgatgg gaagccttac tgataacata 17700 acagtcaaca cacattttgt aggctatatg cattatatat ggtattatta caatacagta 17760 aggagataac agaaaatgtt atttaaaaaa ccacaaggaa ggccgggcgc ggtggctcac 17820 gcctgtaatc ccagcacttt gggaggccga ggcgggtgga tcatgaggtc aggagatcga 17880 gaccatcctg gctaacaagg tgaaaccccg tctctactaa aaatacaaaa aaaaaaatta 17940 gccgggcgcg gtggcgggcg cctgtagtcc cagctactcg ggaggctgag gcaggagaat 18000 ggcgtgaacc cgggaagcgg agcttgcagt gagccgagat tgcgccactg cagtccgcag 18060 tcctgcctgg gcgacagagc gagactccgt ctcaaaaaaa aaaaaaaaaa aaaaaaaccc 18120 caaggaagag aaaatagatt tactattcat taagtgggag tgggtcatca taagtcttca 18180 tcctcctcat cttcacattg agtaggctga ggaggaagag gaggaggaag aggaggggtt 18240 ggccttattc tctcgggagt gacagaggtg gaagatccac ttatacttat ccatgtataa 18300 gtggatctgt gcagttcaaa cttgtgtttt gcaagggtca actgtataca cacacataat 18360 atacaggata cacatataaa ttttatgtgt atatataata tgtatacata cacttttctc 18420 ctgagatttg aggtccaaaa ctgaaatacc cacatccaca tagtatttgg cacacaccat 18480 atacccagca aatgatattc ctgtgtaaat ccatacctat cttttcattt attttatatt 18540 caccaattgt caagtaatag agcattacag tggagtttag gaaacttgat ttttggttct 18600 gctttctgcc attctgttat tttcaactgg cagcttgggt ttcttcatct ctaaaataaa 18660 agggttggat aaaatgatac ccgaggtccc tttcagctct aaaagttgat tctttgattc 18720 tcttgtgtac ctcactgtgt gaggtcccat gggaggttcc aaaaggactc tgctctcagg 18780 gagtttacaa tatacttggt gaatggcctt ggagctctca tagcctaggt tgaggtcagc 18840 aaatgtcctc tgtaaagagt cacagggtga atgtcttaga ccttaaaggc tatacagtct 18900 ctgttgcagc tacccagtta tgcctttgta gtataaaagc agctgtagac aacatgtaaa 18960 caaatggaca tacctgtgtt ctagtaacac ctcatctaca aaaacaggtg tgagctgatc 19020 gctgacctag gtacccttcc aagaaggaat tgaacgaatg tggcatgatg cctggagctg 19080 ggggcttggc ctttgctgta aatggttatg aggttggagg agctgccttc agtgagagtg 19140 gccatattca cctgtcactg tccattccat gaagcactga gatctggcaa cctcagggtt 19200 gagctgtaaa atgaagcatt ctgaattact tggagaacat tctgatgtga atccaggtta 19260 ttaatattcc tatcctgcat gtgttgaaat attcacaggg gctgaagctt aggactttgt 19320 tgccaggtcc atagaaaggt ttttgtggtt gcagactcga gtcctcaggt ggaagccctg 19380 gatgtagaca gaactcattc cctaattcta ccactacgcc tcctctcact tcccctgcac 19440 ttggggtgct attctgacaa atcattccct tgatatttac tgcttagact cctggctcct 19500 gccaagccat gtgctgctga cacatgctgc tcctctgagc agtgtcctaa catctgtgtg 19560 ctgggacata gccacctgcc tatgagaagg tagctaggaa gtttccactt ccctagtggg 19620 aactcacagc aggtctttcc aggatgtcac cgtgtgaaaa atataatgat tagctcattt 19680 ccaaaccaat tttgcatacc aggtgagtta gccaagccat tcaagcagga aagccattct 19740 gtgaagcccc agacaaccgc tcccaaggac agattacctg agaaaggaga atactgtctt 19800 atgaggtcac acattcctaa tggtaaacac tcggggcagt ttctgctctt gactccccta 19860 cacccttgat caaggcactt ggcctctctg gggaattttt caaccatgaa acagatggaa 19920 ccacattcct gggcctgctg ggtcctgggc ttaattcaga ttgtataaac tcatggaatc 19980 tacttatagt ctctggcttt ggttaactta ctttggttaa ctttcacatt agccactttc 20040 attgtgtgtg tgtgcatgcc taatttacct gatgttgcca ggccttttcc atagtctcaa 20100 atgccatgat ctggcaggaa attggttctt tcttttgcca acaagtaaca ataaagggca 20160 cttttgccca ctattcatat gtttgtattt tgggagtatt tttaactgat tttgttattg 20220 atgtatgtag tagatattcg tcctacttca ttgctaccca gtgccatttg aacacgcttc 20280 ttaggcccag catcttcaag gcagaagcca agaactggct ttcttttttt ttttttttct 20340 tttattatta tactttaagt tttagggtac atgtgcacat tgtgtaggtt agttacatat 20400 gtatacatgt gccatgctgg tgcgctgcac ccactaactc atcatctagc attaggtata 20460 tctcccaatg ctatccctcc cccctccccc caccccacaa cagtccccag agtgtgatgt 20520 tccccttcct gtgtccatgt gatctcattg ttcaattccc acctatgagt gagaatatgc 20580 ggtgtttggt tttttgttct tgcgagagtt tactgagaat gatgatttcc agtttcatcc 20640 atgtccctac aaaggacatg aactcatcat tttttatggc tgcatagtat tccatggtgt 20700 atatgtgcca cattttctta atccagtcta tcattgttgg acatttggct tggttccaag 20760 tctttgctat tgtgaataat gctgcaataa acatatgtgt gcatgtgtct ttatagcagc 20820 atgatttata gtcttttggg tatataccca gtaatgggat ggctgggtca aatggtattt 20880 ctagttctag atccctgagg aatcaccaca ctgacttcca caatggttga actagtttac 20940 agtcccacca acaatgtaaa agtgttccta tttctccaca tcctctccag cacctgttgt 21000 ttcctgactt tttaatgatt gccattctaa ctggtgtgag atggtatctc attgtggttt 21060 tgatttgcat ttctctgatg gccagtgatg atgagcattt tttcatgtgt cttttggctg 21120 cataaatgtc ttcttttgag aagtgtctgt tcatatcctt cgcccacttt ttgatggggt 21180 tgtttgtttt tttcctgtaa atttgtttga gttcattgta gattaaaaca agcaatgggg 21240 aaaggattcc ctatttaata aatggtgctg ggaaaactgg ctagccatat gtagaaagct 21300 gaaactggat cccttcccta caccttatac aaaaatcaat tcaagatgga ttaaagactt 21360 aaactttaga cctaaaacca taaaaaccct agaagaaaac ctaggcaata ccattcagga 21420 cataggcatg ggcaaggact tcatgtctaa aacaccaaaa gcaatggcaa caaaagacaa 21480 aattgacaaa tgggatctaa ttaaactaaa gagcttctgc acagcaaaag aaactaccat 21540 cagagtgaac aggcaaccta caaaatggga gaaaattttt gcaacctact catctgacaa 21600 agggctaata tcaagaactg gctttctaac cccacttctc ttttgcttaa tttagctaga 21660 gtgagttccg tggtttgcaa aaaagagccc agatcattat ttctttcatt tctatttttt 21720 tttttttttt agttctgggg tacatgtgca ggatgtgcag gtttgttaca taggtaaaca 21780 tgtgccatgg tggtttgctg cacctatcaa cccattacct gggtatgaag cccagcatgc 21840 attagctatt tttcctaatg ctctctctcc ccccacccca gccctcaaca gtccccagtg 21900 tgtgtgtgtt gttccacttc ctgtgtccat gtgttctcat tattcagctc ccacttatac 21960 gtgagaacat atggcatttg gtcttctgtt tctgtgttag cttgctgagg ataatggctt 22020 tgagcttcca tgtccccgca aaggacatgg tctcattcct ttttatggct gcatagtatt 22080 ccatggtgta tgtgtaccac atttctttat ccagtctcag attattatct ctttagaaaa 22140 aaaataaatg gaattttctt ttgaatctta tagatcctat gatattaatt gtaacaccta 22200 atttgagtgc ttctaaatgt ctcattagga tatttagcac caaagattta ttttacaact 22260 tcagtacatt atttgggatg cttttaatag agaaaacaga aatgtcaacc cagtattgta 22320 ggtagggctt gtatgaacac agaattatct tgagttgctg ggccagcctt gggaggcctg 22380 cgtaaagcag atcacactgt gagttctttc tcctccctct ccatcccctc ttctgttacc 22440 aaattgaagg cttctcactg agtgaaatca agttataaaa atttctgaaa gcctccttca 22500 agggaaaaga ataatagtaa tgacagctaa cattgactaa atgcttacta tgtgtgctct 22560 aatagcttta tgtgaattta ttatttaatc cccacaactc tatgtattaa atactattat 22620 taggattgta tagatgagga aactgaggca ctaatggtta agtggcttgt tcaaggccac 22680 acagcttata catgttccta acctatcatc tctcctaagg agtggccaaa ggggcctgcc 22740 tggtatctgg ggtgaaggaa cagtgttcct ggccaccttg aggttttttt taaaaaaaac 22800 atttctttct ttaattcttt cttcccatcc cttttcctgg acactataat aggcttgaat 22860 attgagaata tactaatagt acttgttcac tgtacatttc ccaagtttat atgaaaattt 22920 aaaaaggaaa aggactgtta tttataaata tattagttcc ctttaattat tttctggtga 22980 gagagtgaat gggtgagggc aagagcagac ataattgtaa ccaactgtca aggataggaa 23040 gtgtcaagga agcttcagca gtggttggag ggaggtcaaa ggtgtaggca ctgggcaccc 23100 tctgtctctg taggtctgtg ggcactgtac acagtggact tcctttactt gcccagattt 23160 atctgcttct ttcaccactc atgagacatg gtgttgggta atacatgccc aattttttat 23220 ttttattttt attttttact ttaagtcccg ggatacacgt gcagaacgtg caggtttgtt 23280 ccataggtat acatgtgcca tggtggtttg ctgcaactat tgacccatcc tctgaattcc 23340 ctcccctcac ccctcaccct gcaacatgcc ctggtgtgcg ttgttcccct ccctgtgtcc 23400 atgtgttttc gttgtttaac tcccacttat gagtgagaac atgtggtgtt tggttttctg 23460 ttcctgtgtt agtttgctga ggacaatggc ttccagcttc atccatgtcc ctgcaaaaga 23520 catgatctca ttcattttta tgactgcata gtattccatg gtgtatatgt accacatttt 23580 ctttatccag tctatcatca acgggtaaat aaatgcccaa tttaatactc atgacattca 23640 ctacctgagc aacaacatgt aagacagtta actgttatcc ttaccctcag cctgttgaat 23700 tcccatctgg tcagccttca ctgaagatgt ggtcttggtg gggatatctg ctgaccatga 23760 gccctgcctt tagtgaacca catggagaaa taattcaact tatttatgtc aatctcagga 23820 aaaaagctga gggctgattt taagatttta agtggagaag ccaactttag cttaacacat 23880 caaagaactt tcttaaaata aaacttcccc aacagagcaa tggggtatct tgagaaaggt 23940 ggtgagcttc ccatgaacgg atgtatttta acagaggctg aatgatttcc gcagggcttg 24000 ctatagaaag aattcatgta tttgggccca acacatagta ctgggcacat agtaagtgta 24060 cactaaccaa tcgcagttgt tagcaaatct gttcccactc tagtggccct tgtttttacc 24120 ttctccttag tttacagttt tatttaaaaa ggaaaggtga aagtggtcag tacctctgtt 24180 atgtgaaagt ttgtcagggt tagaaatgtg attgaatgat gctgtgtttt tgtagtgcta 24240 cagttactca gagtagcaag agaggggggg ttttctctcc catgtcctgt ggttgatgta 24300 actcacacag atttgaggtc ctcagatatt tatctggttt atgttgtaac aaaattggat 24360 tgaagatatt tgaaagcaga tgcattgatc tttacttcca catttcctac aactcttttt 24420 gtaaagctaa gcatttcgag gtgcaaatga gagtttcaga gcttgattga ttgttgaata 24480 aagtcttgcc taccatagtg atcatattta taaaatatta aacatgctaa tggttccctt 24540 ttgtccaaaa tgtattttgc tcagttactt taaaatagat gtgagaactt catagtttgc 24600 aatggcaatg tagacattag aatttcagaa attttcctct tatgaacaat tagatgtttt 24660 ctgctcatat ggccttataa aatctccccc atctctgaag atttggaaaa caggtcatgc 24720 ccagtgtgct tcaggaccaa ctggagtttt ccagttttct aattgctagc atctgtttcc 24780 tacaagaatc actggaccag aactgctttc ttggttattg atgggaaacc tctcctcaaa 24840 tatctcaaaa tgcttttcag acctctgccc agttagcttg tgtttgtgtg tctgtgtgtg 24900 tgtgcacaca caggcacaca tatgcacaca cacacagatg cacatataga ccatgtattc 24960 tcaaggcagg ggggtaatta tcaccccaaa agtaataaaa ttggttcata tggggtacaa 25020 aattacactc tttttatata aggcataaat atatacatac agcccttaaa tagatataca 25080 gtgtatctgt ggtattaagt ctatataagg ggtgattagg gaaaacatgt ttacaaagcg 25140 tccttgaggg aggtgataat gaaaataggg tggagaaaca ctgatacaga cttacagcag 25200 atatactgga gtgtaaatac acaggtattt ctgcattcac atacaggcca cccatgcata 25260 tgatgcatat gtatctgtta ggcataaaca cacgctatgt agtttaccca gggaattgaa 25320 ttcacagtgg ggcaggactg aattctgtgg tctttctaaa attctcttca ataaagaatc 25380 tctccgtgaa ggatttgttt tcactttcag gatccccatt aaaatatttt tttaaaataa 25440 acggtcattc ttattttgac ttgcaggcac ttcaattaac atcacacagt cttcaaacac 25500 agagagtaca cattcgctgt cattctctgg aaaggctgcc tgttagaaat gagctgggtc 25560 aacaattcat ttcactagac atctgctgag catctcctac gtgtggggca tcacagtgta 25620 tcccacagga tgtgcaggaa tgcaggacaa ggtccccaag gcagagtcag atttagaggt 25680 gggctttata ggtgtgagca cctgatacca ttgaagatgg taggttagaa tggcctgaga 25740 gtggggaagg ggcttgcctc acagcctctg gaactcttga tacaactttc tcttccatcg 25800 ctgcccttta tcaaccttat tctaatgccc atctttcctc ttgttaatta tttttgctat 25860 gatcgtaagt tgttttgatt tcgagtgccc ctaaagtctt ttaggaagta gatggtatgc 25920 acaaatccac aacataacaa ttaaagtcta attttttaaa aatcttgtta tggattcaca 25980 cagtttcaac caatagctgt gtttgacaag gctgtcaagt atcagtgttg agctcctcac 26040 attctgtctc attaacctct ctttctacta tagcaaccct ctgctcacac atacccactc 26100 ccagatgtat ctgaggaagc tttcttttcc ttccctcatc cctacattca tcagttctgt 26160 agatgggacg tgaggaaacc ccatcttcct ccaatacaag cctgtctcta ggaaatgatt 26220 tccagaaata ggggtatatt agtttgcttt gctgtgtaac aacttaccac aaatttagca 26280 gtttaaaaca aatacctttc ttatctcaca gtttctacgt gtcaggaatc tgacaatggc 26340 ttagctgggt cctctgctct gtatctcaca ggctacagtc aaggtgtcag ctaggactgc 26400 atccttgtct gtagttctga gtcttcttcc aagctctggt gggtgttggt agaattcagt 26460 ttcttgcagt tttaggattg aggccctcag cccctagagg ctgcctatgg tacagtgcca 26520 actggccctc ttcacaggaa attcacatga tagtagcttg cttttttagg actagcaaga 26580 atctcttttc tagcagcttt cacccagtaa ggccacccag gatagcatct cttttgctta 26640 attcaaaagc aactgattta ggccttgata acatctgcag aaaatctttc ctttactatg 26700 ttctgttagc tagaaacaag ccatagatcc cattcacact cgaagggatt atatataagg 26760 catgaatgcc agaaggcaag gggatctggg ggtcacttta gggtctgtct gctgcaggtg 26820 gcttgagcta atgttcctcc acaataataa cagtagccag atgcttcatc taatcctcac 26880 atggacagct tgaggagaga cttgatatgg gtctatttca catgaaagga acctgagttt 26940 cagagatcca gtcccttttc caagatcaca gagctggtag gagaagaacc cggtcttgaa 27000 tccaggtttg tctgacatca caccaatcaa acaacaacct gtgtgccact taagggagct 27060 gacagtctga aatactccaa ggttatcatt atattgcaaa ctcaagtttt attttgacct 27120 ttctggatgc tcaccaactt tgtttaatct tcctaaattt agattaagag cacatttctg 27180 atcttggact tggcatcagt atattttgta aggcaggatg aacaccctta ctatgtaaca 27240 ggggttttca gaagaaactg cagtgtgaat gttattattt aaatttttgc tgtgaggtaa 27300 ctcacctgca ggcaggctca gagagcttgt gtgatgctac acatttatgg ttgatgggag 27360 tgagatgtga aacccaccat ccttctatca cacttgggtg cttgttggcc aactcaagtg 27420 gttttctaaa acaaaaagga aacaaagagt aaaagtcaag atggaggatt ctttgtttcg 27480 ttcaggcagt tatattttta gctgccccta actcactgac ctattgctgg aatggaaact 27540 agtgacttaa acatgaatgg aaaccacttt aaaaagtaat agggcattgg tgctgcctac 27600 agtgaatcag agggtgaagc attcagatga gtgcaaaaag tctaccttgt tttgtttgat 27660 cttaacaata cctttcattt gtaaaacaca cagtagccta aaaagaatat ttgaataatt 27720 atataattta attgtcctaa caaccctgtt cacatgaccc aactttccag atgaggagaa 27780 gagtttcagg gagaaactcc atgtcttccc caaggttgcc tggttcagag gaaaagcagg 27840 gtcttgtgca tagggattca cttccaagct cctattctac acttccctct caagaacaaa 27900 gatgcatata tggatggaga cgtcgctgga gagagtgcat ggatgtgaga tgaatcccat 27960 catagataca tagaaacacc caggagttag acccagagtt tagccatcaa tgtcctgatg 28020 gtgaagttcc tcaaattgct agcccagggg tggggttttc actattgcct cctattctaa 28080 agggcagata ttattttccc tgcaactctg cccattgggg aattttggta tctttaatgg 28140 gatgcctacc ttgccccagc taacttctgg taaattttaa tattccctat ttacaaaaca 28200 gaaccactgc agagattgct ataaagagca aggtgggcct ttctcacttg accattcaaa 28260 ttaaactata ttacctgact tacgtgctta ttaacgtcat gagtacgatg ttgtaagggc 28320 agcactggag atactggtca aacagtaata ttatcaccat agaaaactgg tgaatctaga 28380 aaactacttc cattgatcta catttcaatt tggggatttt tcttaatgtt tttaccaaaa 28440 tttcaaagca attctgactt ttcctagcca aaatgatatc acctttttta cagagataat 28500 atcaggaatg agaatattta tagagctcta cagcttccac aggatgctga ggctaattgt 28560 cagagaagct ggaaaagggt gtctcaggtc ttagtgatga caatcaggaa aggagacaat 28620 gttctcctaa aacaccacct ggaaagcatc agtgggggtg gggagagtac ttaatgtcag 28680 agtcaatcct gttgtgtgtc tcttcctcct cccaacattt tgacaagagt gaaacaagat 28740 tcaaatggat gtcaaatgca gcctagaaag gcagacagaa tcctgcacat cagcacttcc 28800 aggctacaga gcagttctag ccatgggggc agaagagtca tgctgctacc tgggaagggg 28860 agagccttgg accctaggta ctgaccctct gtattcagaa cccaagcctc atagactgag 28920 actggccttt agctttctcc ccaggtcatt ccccctctgc cagtggctgc agctgcactg 28980 agcctgccag aggtcggagt ctggcagaaa ccccatttgt cttgacctgg aaaggatctg 29040 ccctcctgtg ctcacactat tggctgggcc agcaccacaa agcgcaaggg aggaacttga 29100 gaagtcagca gcagcctttg ttctcaaaat gcttatgctg tgatttcata ctctttctaa 29160 ttttaatcta aacttaacct ttgaaggcaa tttaatagaa ccctgctaaa acaaggataa 29220 tagttaagcg tggcatgaat ctcctcaggt tttttaatgg caagtagcca cagggattca 29280 tgctttggag agatggctgg atcagatgat tcctaaagcc ccttgtgatg gttaattcat 29340 gtcaacttga ctgggccata ggaggcccag atagttgatc aaacattatc ctgagtgtat 29400 ctgtgagctt aacatttgaa ttagtggact gagaaaaaat agattgccct ccctaacgtg 29460 gctgagactt aatcaattga aaacctgact tggcagagtc atgggggcag aactgccacc 29520 ccagcaggct aagaaggtgg gatatcaaac taaaaaggac tattctcaga ccttaagatc 29580 taataaaatt tgccttgcta ggttttggac ttgcttggga cccatcaccc ctctcttctt 29640 tccaatttct cctttttgta atgtttatcc tatgcctatc ccaacattac attttggaag 29700 catgtaatgt gtctgatttc acaggttcac agctgcgaag gaattttgcc tcaggttgag 29760 tcacacctca ggcctcaccc ttacctgata tagatgatat ttagatggga ctttggactt 29820 tagactttag aattgacact ggaatgagtt aagactttta gggctgttgg ggtggaataa 29880 atgcatattg catgtgagaa ggccatgaac tttggggggc cagggacaga atgcagttga 29940 ctaaattgtg ctcaccaatt catatgttga aatcctaatc ctcaatgtga ctgtatctgg 30000 agatagtgct tttaggaaat aattaaggtt aaatgaagtc ataagagtgt agccctaatc 30060 caataggatt gggggattta taacaagagg aagagagata ttctctctct ctctccctct 30120 ccttctccct ctgtctacca agtgaggaca gagccggaag gcagccatct gcacaccagg 30180 aagaaggccc ccacagcaat caaattagcc ggcatcttga tcttggattt cccagcttcc 30240 agaactgtga aaaataatta ttttgttgtt atttaagcca cgcaatctat aggattttgt 30300 tatggcaggc tgaacaggct aagactttgg tgttgtgatt ataacaacga tacaaaaatc 30360 tatcttatag ttccctcaca ggtcaaccct gactgtcagc taacaaatca ataaacattt 30420 tctggatttc atctgtctat aaggcactgt agagaatata gacatgtgta caacatgttt 30480 ctttctccaa tggagaccac agtttagcag aagagataga gcttaggtgt gcacaccaac 30540 tggaatctaa ctttaaaggg cagcagaaaa gcatgctgaa tgttgagttc agagggaggg 30600 gccataactt ctggcaggac aattatcagg aaaggttttg tagagtgaga agtgatgtta 30660 ataagtgtct ctaggaagca aacattttgc agagcttccc acataccaac taagttaatg 30720 gggtcaaatg cttgggttaa gtccacagtg ccactcatgt tgctattctc agcatcttcc 30780 agtggctgcc agtgaaagag tatcaggtct ccagttccat gggcaacaca agaggcacac 30840 tgactttcaa gtgagacaca ctgccactga tacatgtgag tagttcatgg aaaatgactc 30900 aggttaatgc tatcctggaa ttgtgtacaa ataatatgac cccagggtct tctgtccctg 30960 cacaagactc ctttaagttg ggcaaggtca catttactgc ctctttataa tcaaagcaat 31020 taagtgaccc cacaagtggt ttagctacta gctgctgtac ttgtggaatg cagggtctta 31080 ccttaccagg gtctccaccc agtgaatgag cacagaggaa atctcagctc tgaaaaccaa 31140 acaggacttc ataacttcat tgagcccagt tgttttcaaa tgtttcctgg tggcaaggaa 31200 ttctttgtta tgtcaactaa atcatgtgtg gagcctcact atagaaaaaa aagtttaaag 31260 aaggccttct ctggcttatc ctggctgtgt ccttcttgac cccaaagtgg cccttaaggc 31320 accctcacaa aaagctggag cagacaattc taccctttcc atttgcagac atcactacct 31380 tcttaggcta caatgtcctt tcatcagaat gaatgcatgg caggattttt acctgcctga 31440 ggtcacacag ctaatcattg gccaagctgg gagtagaagc tgggtttcct gactcctaac 31500 ctagtgctcc tcaccatctg ggtagcttta ttcaatggta ccaaggattc tgaccaccct 31560 gcattcctgt tacaccaaag aatccagtga tgctggggaa atcaatgatg gtgtgtcagg 31620 atctcagggc atgatcaggg ttaaaggcac tgtgttagtc tgttctcatg ctgctaataa 31680 agacataccc aagactgagt aatttataaa ggaaagaggt ttaattgact cacagttcca 31740 catggctggg gaggcctcac aatcatggtg gaaggtgaat gaggagcatg gtcatgtctt 31800 acatggtggc aggcaagaca gagcttgtgt aggggaactc ccctctataa aactatcaga 31860 tatcctaaga cttatttact agcacgggaa agacctaccc ccatgattca attacctccc 31920 actgggtccc tgccatgaca ggtgggaatt atgagagcta caattcaaga tgagatttgg 31980 gtgggtacac agccaaacca catcaggcac caacaagaga taattgaggc ctatacatgg 32040 gagaacaact acaaggaccc tttacttaga tctgaccctt tacccaaaaa gagtcactgg 32100 aaggaggatg acctttggaa aagggatgca gccaacccag agtggcctgg tagggatggg 32160 gccaaggcaa taaataccca acttcgcgtg cattaggact tctgatccta atgcagccag 32220 agggcaggga gccccttgat gctattccca ctgtcagcct ccaggcccca agcagagtgg 32280 tgaggctaga gagcaggtct ggagggacac agaggcattt ccaacccagc tgctaagaaa 32340 ggagtcactt cactgttgca ctgcatcgag cttattgaaa cacagtgttt tggggatgag 32400 cctgcttttt acagaaaggt acctatggga agaaattaac aaggaggtca aatacacata 32460 cttcctgctc tgctgtacta aatggcacag caacttttag attctcgcct gaaaattaga 32520 gtatatgaga ttaaaccctg aaggtagaat gaattgcttc atgatctgct ccctgttatc 32580 tctctgggcc catctcttac cactccctac ctcactgccc aagtgacaac cacactgaac 32640 aacagctcag ggctcccaga ggggccaggc tctttcttct ctcccacagg tttccctctc 32700 tatgtggatg ttccttaccc cctttgccag ggcttagctt ctagaaggcc tttccaggcc 32760 ctccctccag catgaccaag ctaatttcag tgtctctctt gggtgttccc acatctccca 32820 tgcacttttt cactgctcag atcccattgc cttataagag tatgagtgtg ccaaatgttc 32880 tgtgtcttcc ccgtgaggca gctatgacgt caaggatggc agttaccgac cttgtttccc 32940 tggtattaag acagttccag gcatagagct gctcgatgaa tgtctttgga gggaaagaag 33000 gatttcttga gacccagtca tctcatcttt aaaaagaaag aaaacatcat ggaaacctca 33060 atgaattctt cattagtgat tttgaatgga tggctgtaaa caaagctgct cgctgaatgg 33120 cttatgtttt tcctttaaag tccaccatca gaactccatt ttgtgggtaa ctagccccag 33180 cgaaggcaag aagaccatga aagccttgcc ctgcagtgac caggggagag catcctggtg 33240 acctgtcatt tgcaaacatc tgtatgttgt ttcaaagttt atgaagcaca cgtactacat 33300 tacctagaac ctcaaaacaa aactgtggag gcggggcact tagtacaagt gtcattctca 33360 ttttactggt agagaaaagc tgaagctccc aaagtcaact gtctcgtctc aaagccacac 33420 agccgggtag aagtcaatgt caagctgggt tccggagcac ctgtcaagtg tcccttccac 33480 cccaacatgg tgagtccagg gatagcacag gtgacagtac tggcattgcg gacctgaaga 33540 cagcagactc ccaggccagg aggcctggaa agtactcgag aattaattct acacatccct 33600 ctcatcttga gcacaaggaa ccaatgcact gatggctcct ctaatgtttt atttgtcttt 33660 taatcccaag tactttttta aatctctagg ggaaaaccaa ttcaattttt gtactagttt 33720 tctctttcct ttactatgtt ccactaagtg ctttcccaca ctctctttga ctcttttctt 33780 gaagggccta ggttctattc atctctgtga tgatgttttc tttcaaaaaa tggtttttct 33840 gaggccctac tatgtgctgg acattgattt tagtgctggg actacagcag tgaacaaaag 33900 agatatggtt cctgacttaa ggagtcttgt gttctagtgg atgagacaga gaagatataa 33960 agaaataagt caagtaattt caggtgataa aaagtgctat taaggaaata actggaggac 34020 taatttaaat gggagaaggg tataaaagac ttatctgaga aaggcattat atttcaggcc 34080 cgaaggcagg ttcttagccc ttaccaggtg aggaaatatt tggtgattga atggatgtgt 34140 gcatacattt aaatgaatga atgaatgaag ggttgtctat attctctctt tacacaggca 34200 ttcccccaac ctaccttgtc ctacctgttg cctacatgcc agccagtcca tacctgaatc 34260 ttctatgtgt cttctcagag atcccaagcg tctgtaactc ctaaggccgt gatatgcagg 34320 agcagggtag ccagagcgga gctataagga atgccacaat cgctcttcca accgattgtt 34380 acaaaactct tctcaatggt gtgtcttgtg ccaagcattc ctcttcctgt cctgagggct 34440 gtatattgac atcacttacc caaacccaac tcccactagg tagtataggg acactactca 34500 tttgatgaca ctttggtggc tctgtgcatg acagcacaca ccctgtccca gaatatatgc 34560 atttttatgg cttcctagta agaatactat ccaagtcctc cccagcattc ctctcaccct 34620 ccctccctgt gctgctgcat gaactttaga tcaggtgagg tcaggcagtg aaaaagtccc 34680 tgggcagagg tctgaggatt taacagattt gttgagcatt atgatctctc actgaatgta 34740 cgttacaagg aagcccacct cctttgtttt attggcattt cattttcttc catttcccag 34800 accttgtggc cttttccctg tgcagctcat aaaagtaggc aaccagcctt agtaattcac 34860 cttatgcttg aatcgtttct caccttttac caaggcttat tccatttcta gatttctcat 34920 tattcttttc agctcttaca aactccaaat tcatgccatt tagactgatt cccagaaaag 34980 agataaaagt gtcagtcagt actaaaagcg aatcattgac ttcttaaatg tcccttatta 35040 aaaccactca tctatgcagc catgcaatct gagacaaaca gagttgaccc aagtctttat 35100 ctccatgtgt aaaatgtctc atgagttgca gagtcacata ccacataata acgtttcagc 35160 caacgacgga ccccgtagaa gacggtggtc ccataaaatt ataatactgt atttttatta 35220 tgctttttct atattttgat acacaaacac ttacccttgt gttacagttg cctacggtgt 35280 tcaatatagc aacatgctgg acaggtttat agcctaggaa caatagacta cactatataa 35340 cctaggtgtg tagtaggctg gaactatcta ggtttctgta agtacactgt atgatgtttg 35400 ctcagtgaca aaatcaccta aagatccctt tctcagaatg catccctatt attaagcaat 35460 tcgtgactgt ataaaaaaat ttataatatc atgaactcat ccactgaaag gactggaaat 35520 gccttagaga tgaacaagct tgattcatga ggacactgag tcccaagcag gagcagcaag 35580 gacaggcccc aggtgaccca aactgataag atggagtcag gacaagaatc acagcccctg 35640 catggttagg tgtccaaggt ttgttcacct ttccaaacta caggttctac agctacctct 35700 tcttacacag aaaccttccc ctaaagctga gctccctcac atttcccgtt tctcatattg 35760 tctttgccat cttccaacat tccaagatca aaacctcagt gtcatccttg actgtcccct 35820 atatttcttt ctttttttct tttttttttt tgagatggag tctccctcta tcacccaggc 35880 tggagtacaa tgctgcaatc tcagctcact gcaacctctg cctcccaggt tcaagagatt 35940 ctcctgcctc agcctcccga gtaactggga ttacaggcac ctgccatcat gcccggataa 36000 ttttttgtat ttttgtagag atggggtttc atcatgttgg ccatgctgat cttgatcctc 36060 aggtgatcca ccctccttgg cctcccaaaa tgctgggatt attggcatga gccaccgcac 36120 ctggcctgtc ccctgtattt catcagacag agagtcttcc tccagagtgg ttcccttggt 36180 agatgcccac caaaactgaa cagcaatatg aaagtgcaag gttttcctcc cacctaggcc 36240 tggcccccac agaggtggta actaagggag tccacattgt atctcattga gtatcaactg 36300 cttacagaaa aggaatgtaa tatccattat ggtctttgat aaatatataa atcagatttg 36360 cccactgcaa agcacaagcc ttgagaaacc acattacttt ttgaggtata tttagaagct 36420 agttgtttaa ttttcttgga acttacatat cacttacttc tggctcctta tttagtttgt 36480 aaaaagttca ggatgtttca aaatttcaga aattatataa gtagatacta ctcatctagt 36540 aagcacgtgt aaaatgaaat agcaataaga cagaaaatga ggccaggtgc ggtggctcat 36600 gcctgtaatt ccaacacttt gggaggctga ggtgggtgga ttgcttgagc tcaggtttca 36660 agaccagcct gggaaacatg gaaaaaccac atctctacaa gaaatacaaa aattagccag 36720 acctggtggt aaatattgta gtttcagcta ctcaggaggc tgaggtggga agatcgcttg 36780 agccctggag gttgaggctg cagtgagccg agatcacacc actgcacttc agcctgggct 36840 acagagagag atcttgtctc aaaacaaaac aaaacagaaa atgaaaataa gcactaaatg 36900 aacctgtaat aagggtcaat gcatgcttca aataatgatc tttgagcttc taagaaggca 36960 aggagggaaa aaagacacaa tgagttatag aattctcttt ttgagagtgg aagaagacaa 37020 tttcttggag aagatgaaat tccctgtcat tgaattactt tgaaaaatat ttttttctat 37080 ttataaaagt aattagataa cttctgataa tgttttggtt tctttctagg tttttttttt 37140 ttaattattg gaattataca accgatacat tttgcagcat gctctattca caacatttta 37200 tcactagcat tttcccttaa cattaagtat tcttttcttt gtggtgatag attctaaagc 37260 catctctctc ctttttcaac ttttattttt gttttggggg tacatgtgct ggtttgttac 37320 atgggtaaat tccatgtcgc tgaggtttgg tatataaatg atcaccgtca cccaggtagt 37380 gagcatagta cacaataggt agttttccaa tgctcacccc actcccactc taccccctct 37440 agtagtccct agtgtctatt gttcccatct ttatggccat gtgtattcaa tgtttagttc 37500 ccacttataa gtaagaacat gcagtatttg gttttatgtt cctgccttta tttgctacct 37560 gaaacagcat ggtactggca taaaaacaga cacatagacc agtgaaacag gataaagaac 37620 ccagaaataa agccccatgc ctaaggccag atcttcagca aggttgacaa taacaagcaa 37680 tggagaaagg actccctgtt caataaacag tgctgggata acaggctggc catatgcaga 37740 agattgaaac tcaaccccta cctttcacca catacaaaaa ttaactcaag atggattaaa 37800 gacttaactg ttagacctaa aactataaaa ttcctagaag aaaacctagg aaatgccatt 37860 ctagacatca accttggcaa aaaaaaaaaa aaaaaaaagt gactaagtct ccaaaagcaa 37920 ttgcaacaga aataaaaaat tgataaatgg aacctggtta aactaaaaga acctctgcat 37980 agcaaaagaa actatcaaca gagagagtaa acagacaacc tacagaacgg gagaaaatat 38040 ttgcatacta tgatgcatct cacaaaggtc taaaaggcca tttctcatgt aatggtttat 38100 aagtagtgtg tggggatcac tcaaaaatat aatgaacaat gccatcaacg ttttttaaat 38160 agaaaatgca gtagaaattt tgggtgatta tttcatctag taactgtaaa aactgaggcc 38220 agggcattaa aatgtgattc aaggagaatg atttttcaca ggtgctaatc ttatgcagta 38280 ggaatatcta atcttgtcag cccaatgtga ttcaggacaa ggcagaaggc tggaagagtg 38340 ggtggagccc acatagcctt agtggacctt ccctaaatgt cacttcccag agacaggctc 38400 ttaattcaaa gctgctttaa ggttttgagt taccagggaa tctattcagc tagttcagtc 38460 tttaagatat tgaacaatgc cttagtgaat gaggaaagaa cgtgacatgc ttagagggtc 38520 tcttgtctat tcaaggagtt actttggact ggagaaaggg gaggaaatgc cccttcccct 38580 gaactttgaa gattggtctt cagttagcac atataataca tgtgtcctgg gtcttcatgg 38640 cactgtgaga tataaaggga agattcttat tctaacattt ttgaataagg aaaacaaagc 38700 tcagaggagg taaatgactt gtccaaggtt atatcccagt gaataatgga accagggctc 38760 cacttagtgt tgtctaacct ggtccgtggc tgttgtgtat cccaccctgt tgtgcatttt 38820 ttttcaaact ctggctgttg agctcctttg gaaggctgat ccacagctct ctcataatta 38880 ttttctgttt tttttttttt taatggagtt ttgctcttgt tgcccaggct ggagtgcaat 38940 ggtgcaatct tggctcactg caacctccgc atcctgggtt caagccattc tcctacctca 39000 gcctcccaag tagctgggat taaaggcatg tgccaccatg cccggctaat gtttgtattt 39060 ttagtagaga tgggatttca ctatgtgggt cagctggact cctgacttca ggtgatccac 39120 tcgcctcagc ctcccaaagt actgggatta caggcatgag ccactgcgcc cagcctcata 39180 attattttca atcccaactt ctagtacaga tgttctccaa gtgtggaccc tacactagaa 39240 gcctcagcat cacttgtaaa agtgttagac atgcaaattc ttgggcccca ccctggacct 39300 actgtgtttt aacaaggctt tcaaatggtt ctgatgcatg ctaacattgg agaaccactg 39360 ttctagaagt tcttaataaa tattgttgaa tacttcctag aatccccata gaccttcact 39420 gaaatattta aaatatttaa gaatccccac atatattttt attggaaagc ttacgattcc 39480 tgctcaggaa tcactcattg ttagccatcc ccagtcaaag agaagagtat tttatcacac 39540 aatgacaagg aagcccaaca ctaagttctc ggatatggaa agcactgagg ggaaaaaagg 39600 caccatagat gggtcagacc aggtataaga tcataatgta atgctgttta ttgagcagtt 39660 attttgcacc acaaactatc cctaaatgct ttacatggac aataaattca caccacaact 39720 ttttaagtac cacaccatat ttctacttta ctgaggagca aactgaagcc aggaaacttt 39780 cccaagatta caaccctagt aagtggcaaa gcctagtcag acccaaagcc caggagcttc 39840 cctgctattg tcttttgtct tcttgctctg ttgcttccag agaaaaaccc caccactgca 39900 gcaagctaca gggaaagatg ctgagtggaa acccactggg catcccttgc acctagaaca 39960 gcaccccaca cattgttgat gtttagtatt tgtcaagtga atgaatgaaa tgaactatac 40020 acaacataat tctaatagaa gtagtctgaa gaggatttcc agggaagatg gggtctgagt 40080 tagtatttga aagtagagac agataatgct gtccaaaaga gagaaatgac atgaacaaaa 40140 gtgcagacat agccaacctg agcctagtca gaggaggtgg gcaagctgtt tgcctagagg 40200 ggaggggagg caggggaaga ggtcttgaaa ggggagaatt tataaccctg gaaaaagaaa 40260 gggtccctga gaggcagctc aaggagtttg aattttatcc tattattagt gcaaagtcat 40320 tggagggttc tgagtatggg ctcaaaaggg ccaaagaaat aatttaggat attgtatcat 40380 atccattttt ttatttttaa tttacaaaag atggagggaa ggtagctggg ctccagatct 40440 ggggggtttg ttgaaatagc aactaaaaga tcatctttca aaaaaagcca cataccattt 40500 tgattcatta caatttgaat gtagcaagaa taattacaat aagcttgtca ttaaagcttg 40560 cactggaatt tgataaaaag aggtttatgt ttcactataa acacctgaag tggagtcata 40620 ggaaaaagca tgcaaaatcc aaaaggccac aaagtcaaaa ataaagaatg aaaaagttac 40680 tataatcctt gttattcaag ggtgaaataa aaataatcac ccaatctgtt tatttaaaat 40740 aaatatttat tacaaatatt tattataaat caactatata gtttaaacac cactttcttg 40800 caaaagacaa agccctgaaa ggaaataatt tattcttttg atatgtgggc agatctgtat 40860 gaactacatt gctcatttag caatgaaaag tcaaccaaac atgggaagac aatatgcaca 40920 ttgcatacat acagcacaca cgtaacatcc agccctaaaa agcatcagca atatcccttt 40980 tgttgttgtt agtgttattt tccgacaagc tgccttttgt ctattctagt taatgttagg 41040 aaacctcata ggtctatgcc attataatgt ccattaccca tttgaatatt tagacagagt 41100 ctggagtgat tattaatact gagaatcact atgtttgatc cagtgaagga caaaactgta 41160 gtacgatgaa ccaaaagtac taagaagggg ttatttgaca tttttaactg ctttgaattg 41220 aataaagtct gtctgtgcat gggcattgta tgaatgagtt tgccattaaa agtaatggca 41280 aaaacagcaa ttacgtttgc accaaccttt attaaatgta agcttctatg tctaaccatg 41340 ccttcaaatt cttttctgaa agtagaaatg catgtacata gagtgaccaa ctcatcccag 41400 ttttccaggg accttcccag ttttaaatcc tgaaagtcct ggaattcccc cagtcctagg 41460 caagctggga tgtctactca cactacacat ttaacaaaga aagaaacaag cagacaaaaa 41520 acaaatctac caagaaaata ataaaaatga aaggacactt ttccgatgtc ccacccagta 41580 gtaaacgctg cttcccaaga catgaaaaac atattgttga gtctcctatt taccatttat 41640 tccattctgc cgtatacgta atataattat tatattaacc ttcatctctc cataggtgac 41700 tggaagctat aatcacttcc actgatcaga gcccttctat gcacatggtc tcatttgacc 41760 cttgctgtca ctctttgtga taggcagaga aggtgtcaga ggcccagaga tgagcagccc 41820 ccacttctta ggggagcatc agcacaggaa cccaggattt ctgattccca gtctggtggt 41880 tttttttcca tcactgatca tttacattct ctttatgaca ttcaggccct ggtcctttac 41940 ctatataaca tagagaaata ggctatttat aatttacaac ataaattaag ccaaagactt 42000 ttaagcactt tttaattata ttgtatacct ttcaagtgag ggtgatgaat ggtactgtac 42060 ccctatttca tagtaagaga aatgaaggct ccagctaact tggctcaagt tgtacagcag 42120 ttgcacctta gagattgaca ggtaacatat tgtcttgttc tttgtaatct gatatacttg 42180 gatttaaagc acatttcaag atttttttta agattagcaa cataactata tacatatata 42240 tttaaaataa atgtccatat tgatagagac ctagacagaa acaagcactt gcctgcccca 42300 atgagaatct gagggcacca ctggttcctg gcacactgga ctgaggatgc catctataca 42360 tgtgttggac agtcactgtt ggccaaagtt aaaatataca ttttaacttt gacagaaata 42420 cttttgtttc agttgcatat cgaatgacag gagccacgtg ggcatcactt taggatctgg 42480 ttactccttg gcacctaagt tactttctat ttcattaaaa gtctttcttt catctttatt 42540 tgccattctc ctgtatctgc tacatggcag gctagtgggc ttgaatttgc tggggtttcc 42600 agtccccagg tcaggccgac ccctgagtgt gcctcaccaa agctacatcc tgctcactct 42660 ccagttctaa atcagagcct ttggaggcac aagtaaaggg acccaagggc agatcagagg 42720 ggttttcctc ttcatatatt ctttcttttt ttgtcacctc ccttttagct tactttattt 42780 gttttaaaaa tacaagatga ctgtgacaac tcaaacctta aagaaatata taggtctaca 42840 attctttatc tgtaatcctg aaattcaaaa agctctgaaa actgtaagtt ctttcataag 42900 tttgtggaaa ttcactttga ggcaaaactt ccttgaactt tagagatgat ttgtagtcca 42960 tctttgcccc acttggtgct gtggagatgt gattataaca gacttgatta cagcagtgtg 43020 gtgtcccaga gcctgcaggg ggtggggatg gggttacata tatatccaca catgttacct 43080 ttctacattc caaaatattt tgaattttga aacacatctg ttcccaaggg tttcaaatat 43140 ttctttgctt gaaaccacat aaagaaaata tgaagactct cctttcacac accccttgaa 43200 tgcacccacc tgaagaaact gatgttatca gtatcaggtc ctgccctcaa ctcctctcta 43260 tggcatgccc tctctctctc tctttctctc tctctctctc tctctctctc tctctatata 43320 tatatatata tatatatata tatatatata tacacacaca cacacagtag taaacactgc 43380 ttcccaagac atgaaaaaca tattgttgag tctcctattt accatttatt tcattctgcc 43440 ctatatataa atataattgt tatattaacc ttcatctttc cataggtgcc tggaagctat 43500 aatcacttcc cactggtcag agcccttata tgcacatggt ctcatttgac tcttgctgtc 43560 actctgactg ataggcagag aaggtgtcag aggcccagag atgagcagcc cccacttctc 43620 aggggagcat aaggacagaa acccaggatt tctgattccc agcctggtgg ttttatatat 43680 aaaactatat atatatctcc tctctctcac acgtacaata cgaacacatg caagattttg 43740 tttttaaata caaaaaggga tcacactgtt cttattactt tgtcatttgc ctttttctcc 43800 tactgtgaca ctccctctag aataatacat aagaatcaaa ctcatgcttt taaatagctg 43860 tatactattc tatcatatgg ttatatcaca ttgtaatcaa ctattctgct tttttttttt 43920 ttgagatgga gtcttgctct gttgccaggc tggagtgcag tggcgccatc tcggctcact 43980 gcaacctctg cctcctgggt tcaagtgatt ctcctgcctc agcctcccaa gtagctgaga 44040 ctacaggcat gtgccaccac ccccagctaa ttttttagta gagatggggt ttaaccatgt 44100 tgtccaggat ggtctcaatc tcttgatctc atgatccgcc cgcctcggcc tcccaaagtg 44160 ctgggattac aggcatgggc cactgtgccc ggccacctat tctgctattg atgaacatgt 44220 tttcatagta ttttgctttt gaggatcaaa tattagtgat tatattgtaa aaggaatata 44280 tatctacagg gtttaattca ttttatatat attgggatgt tggttggcaa aaacaaacaa 44340 acaaacaaaa acacccacaa aaaaacacat ttctctctgt catacaaata gaaaccctga 44400 gacaaagggt gttcatttta aaaaatgaaa agaaagagtt agctgcaagc gagttatttt 44460 tcctgcaaca ggcaggcttt ctatgaacta gaggttgcac agaggcagac tgctaagcca 44520 ctgaactgct cagccatggg catcaggagc tctcaggaca gcacctttag agagcccagt 44580 ttactaaccc agccatgtca tgtttccata acttaacccc tggaggtagg gtccgacatc 44640 tgactggtcc cacaggcaaa gaggcaggta acttcagggg gtgcaggatg gggcctgttc 44700 gagatgaaca tgctcagtga ctctaggttt ggttggcttc taacagacaa gccaagtcat 44760 ggaatttcaa taaataattt aacctcagct ccagtttcag cttcagtgtc cactttcagc 44820 acctggctgc cattgttatt tgcaagcagg ctcctcctct gcacgaggta tggaaatttg 44880 gcagagattc actgacattt ttagaaccca gtagggccag attactatga acaagagtgg 44940 agttttgttt ttgtttttct ttttgttttt cctaaccaag aattatgtga aatctggatg 45000 tgcaaagatg cactttttaa atgaaaaaga aaagctggct atcactgacc caatgcatca 45060 gttttaaagg tcttccgatt gttgctgtat gaggccttta tcttgcaaag gttctgaaga 45120 ccagccttct caaacactaa gaggcataaa tcacttagag gtcttactga aaatgcaggt 45180 tctgattcag taagtatggg gctggcctga gaatcttcat ttctaatgag ctcctaaagg 45240 atgctgatgt tgctggtctg tggacctact ttgagcagca aggctgcaga caacccagaa 45300 tttcctccta acaagaaaac tatccctctt agggcctttt ctgggagttc caggggagag 45360 ataaaaagct cttaatgaga cttgattgag agataaactt acaagaggac cctggagctg 45420 taggacattg tgttctagct ggaatttcaa tccactaaca aagaagacag gatatggcag 45480 tggtaggttc cattattgta tgtcatcctt tcagagcacc tttgatcctc acagagaact 45540 cgtgaagtag gtgggcagct gttattattc ccaattacag ggatggagac agctttgagg 45600 ccaattcaag ttatctttgt tttttaaatt attttctggt tgaaaagata gagagaaaac 45660 acatcttgct gaaagctaat cacataattg caagcgctat acagtaaaag tccttgatac 45720 ccatactcta cttatgttaa atttgtttct aagccttgtt tctgttctgc cctttggtat 45780 gtctctgaga cactggtctt ttcatgcagg ctacattttg aacaactaga gtaaatgtgt 45840 ctggccctga actgtatcat aggactgaga agagaaaagt aaaacaggat ctgttttaca 45900 acttagtaga agaaactcat gttacagaaa ggtccataag agctaactag gaggacatag 45960 gggaaaatta tgcttctcat tttagctttg ttcttatctt ttaattcttc agcaggcctc 46020 ttctctgtgt agaagagtca gcagaattgc atctaaggtt gactttataa ataaaacatg 46080 tcatcaccaa ataatgctta cattctgtcc tacgaatcct agttgagacc tcagaatttt 46140 ctatattatg tttccagcag cctccttcaa taattggcag atgaagcaca agttgcaatg 46200 tttctttgcc attcctgagg ctccatcaaa aaacaatttg aaggggctga tcctaaacat 46260 tagataggat ctagaagact atctgtcatt aatgctcgaa gttcatcacg tagcatcatc 46320 tgttctaaac atagtccaac aatagcaggc ttccttcctt aggtgggtct tgtacgtaaa 46380 ggaaaggttg ggactttggc tcaccctggc ttaagagggt agaaatagtt tggtgggggt 46440 tggggtggag acgagatgac cataataaaa tttttttaaa aacatgtaca acaaccctgt 46500 tcggcacctt caaactaaat aagatataag tgatatgctt tgttgattgt caacgattac 46560 atatacttct actctagacc tatggatgga acaatctctg ctagaactgg aaatgcattt 46620 tcacatattc tatataccag atggctgatt tgaggaatcc actctgtgga tagtagtatt 46680 agaatgtggt acccaaaatg tagttctttt tgtgcttgtg aatttgaatg ccccaagagc 46740 atcatagcaa attctaagac ttacagaaag gatccagtag aggaaaaaga acaatttctg 46800 tacctcattt attttggagt ccaccaatgg ggcagttttg tatttttata atggccactt 46860 tcagtttttt ctcctagaca taaagtggag aatttacatt agagcttttc ataagcaggg 46920 atcacctctg actcctttgt gcccaatgaa caagtgagca taaagagaat cagagaattc 46980 agatgtaggc attgtctagt tgtcccttca tttcataggt aagggtgcag aatgccagat 47040 cgagacagtg acttgctcaa gggcacattc atcttgcaga acaactggga ccagtatgca 47100 ggttgtctga cttccaccag tcaggcagaa aagcagggtg ggcaatctgt ctcttgtaaa 47160 tgcaaaggaa cttgtgagag aaaatatggt agggcttgga aaaattccct ttttgaaaat 47220 aaccactgac tccttcatct caggttgtgg cttcctgggt gagggcctgg actattgtgt 47280 gtgtgtggta taattactgg gtaggtttat ttttttatgg gagtacaata aagagaacat 47340 ttcctgcctt actctgccat ttgaaaaaga ttttcatttc cccttttggc ctggggatta 47400 caaaagacag actcctttcc aattacagct cggtctgagc ttgaacatat aactcagcca 47460 gcagcaagtt caaattgatt ttttttcaag gctgagaaaa cgaaattggg tcttcttgac 47520 acaaaaactg gcttggtttt tttctgtaat tgtgaggctg ttttcaaagc tcccttctaa 47580 agcagaagat ataatcatcc aggactaacc acagtcttca ctgtaagttc tataggggct 47640 cagaagaaat tagttgggtg ggcccacatg acaggggcac ttggctggga gaaacatttc 47700 cctgttgcac agtggcagag aaggactttc tccagacctg tgacctttaa tgtgctggct 47760 gggggtgagg agagggagga aggaggaatg ggcccagagg gcagagtgac cttgaagatg 47820 gcagatgcac tgagtgcttt ctgccaggca ctgcgccccc attttacttg tgttatctcc 47880 cctaatcctc ccaatcaccc taggcgggag aagaaggaac atagtgagaa aagacagcta 47940 cacagcttcc cttctatcct tcccctggga gctcaggagc tgtcgtagga gtgtggcttt 48000 ctgtctttac cctgagtaaa tcctccacga ttaaaaaaaa aaaaaaaaaa aaaaaccttg 48060 atgtgaatgt gaatccatac cattcaataa caaggcagga tttttcttcc tctgtcttca 48120 gcagtttact cttttaaggg aataaaatga aagataagca agggttagca ggaacatctc 48180 tcgaagatga ctttccaaag gattcagttc tgtttttcct gtcttccctg attatgtcta 48240 aattacatac gttcacgatt ttgactgaga tttcagccat ctccattcaa tagcaaactc 48300 catccctacc gatcgtctat gtggccttgg aaaagtcatc tgaagtcttt ggatctcaac 48360 tgccctagtc tgaaggagtt ggatagagca ggagggggtg ggggcgggga catggggtgg 48420 ggacagcctt ccaaaaaggg tgcttcttca actgcttttt aaggtaaggt gctgagaata 48480 ctatggaaag atgctacaga gaagttgtga gaaggcagag cccagaagca gctagcaaag 48540 gtggtcacag gtggttcttc ctttgtggcc tgattttcct tgcaatctac agtgctggcc 48600 cctcccctgc agatccagct gcctgtgttt tcagaataga atgctagtcc acaagctctt 48660 tcatcctctc ggcaaacatt tatccagccc gtgctgtgag ccatgcatgg tgctgggaga 48720 gaggactgta gtagcttgtg gagtctctac tcagccattg gaatttttag gtgcaaataa 48780 aagatttagt tattttcacc ttctcctagc cacattttag gaacagagtg tctttcaagc 48840 ccatcttttc taatttattc actttaaaaa taacaagatt aagacccaga gagggaaaat 48900 tacttgcccc aggtcacaca actctttgtg ccaaaactag gtctggaacg cagaactctt 48960 aatcttctgc tttttattac attgccccaa attgcttaaa gggctaccca gggaattcag 49020 ttcaccaggg aacatcttgc ttctccaaac attcttgcta acaaagatct tactgcacaa 49080 gtataggaag atgtcactat tacaaattca tagcactcat attaacggca aagctaaaac 49140 attcctatag tcacccgcct ccaattaatt aaaaaggaat gtgttgtttt tgaagtcttg 49200 cccttccaac cagccttgaa gtaattttaa atagaaaatt ttaaatggaa attaaggcag 49260 ttttgtgata agagtgcctg ccctttggtc tggggcatcc attgtgtgag ctagtttttc 49320 agagctgcag aagtaacaag ttggcaggat ctcacatagg agataataga taaaaaccta 49380 catgtctggt ttcaggaccc ccaggaagct attgttgccn cttcttcctt ctttcccagt 49440 tctttaatca gtttctggca gacacctgca accccactat tgaagcttca tctcgttgct 49500 tacatctggg ccatgataaa taaaagtacc ccaggaggaa taagtgctta gcagttacca 49560 acaattcata cttaaatgtg aatgctttca gagttggcat tgagagatac aagtggggag 49620 aggcatgctg gccaagagtg gctgaggggt ttagagatgc atgatggtgg agatagcaga 49680 ttgggaagac acagagaccc ccagaagatg agcatttgac tgccttatgg ctttgcctgt 49740 gactaggctg gggtcctcag aatggaggaa gaaagggaac tagagaaatt ctgcatgggt 49800 tttcctaata gagcatgaag aagatctgct tggaattccc tgcacaccca ctgatatggt 49860 ttgaatgtgt gtccccgcca aatctcatgt tgaaatgtga ttcccaatat tggaggcagg 49920 gcctggtggg aggtgtttgg atcatggggt tggttccctc ctgactttgt gccctcccca 49980 tggtaatgag tgagttctcg ctctgatagt tcacaggaga gctgtggttt aaaggatcct 50040 ggttcctctc tctctctctc tcttgcccct gcttttgcca tggaaagtgc ctgctcccat 50100 tttgctttcc actatgatca taagcttcct gaggccctaa ccagaagcta agcaaatgct 50160 ggcatggcac ttgtacagcc tgcaggacca tcagccaact aaatctcttt tcaatataaa 50220 ttacccagtc tcaggtattt ctttacagaa cacaaaaaca gcctaataca cctgcattgt 50280 gcaatccagc ctccaggcat gtgcttatgc catcacttct gcctggatgg ccttacctgc 50340 cactttgtat gaataaactc tcttaccttt gaggctcaca ttaggcattg ttgctgcccc 50400 aaatccttcc gtgaaccctt aggtgggctc cttcctgtcc ccactccacc cattctctac 50460 tactactgca cttcagtgat gacctggcca tcatgtcttt accttttgct gcctctgttg 50520 tgtgatagct tctcaagaga gggaccatgt ctctttcatc accacatcac tacatcccca 50580 gtctctggaa ccaagctggc ccttggtaaa gatgcccatc tggctgtgtg gatccaccct 50640 gcccaagtgg ccaagtgcct gcatatagtt gagagtggag atacagcttg ctggtactcc 50700 acgcagccaa gaaagcccaa aacaggacct tatgaggacc ctgcattttt taagaagcca 50760 catttttcac catggctgac tagaggaagg tggctggaga gaacaaatgt ggttgggtac 50820 atatggcctg tggttgacca catgagaact tgagcccagt gactagggtt tctgtacccg 50880 gtcacagcac actctgtcac cctcagggaa gacccacctg gcagaactta agagttctgt 50940 gtggagatgc aaccataaaa atatcagttg gcacgtgaag ttcttggcat tgtttatggc 51000 tgcattgact tgaggttctt cagacatctt gatccaaaat gggctctcca tgccctgaac 51060 aactcaaaat ggtgcacttc tttcagggcc tgtgcaggga tggaaggtga agaagaacag 51120 tgtagaaatg gggtgggaag taggagtggg gccaggatgt gaaggagttt ccagtttagg 51180 taaggaaaaa ggaaacaaaa agaggaggga gaaacaacgt cctaagtgct actgtcggtg 51240 gttggtccag cccgtcacac atctcctgag cccagagact ctgatgctgc aagtctacag 51300 tgaggtcagg gagctcattt gtttctttgt ttgtttaaca agctcctttg gtgattctgt 51360 tggacatctc ccactgtggg caccactagg tcagaagttg tcagtcctga ctgaacatta 51420 gaatcacctc gagaactttg agggaaaaga attctgatgt ctgagcccta cccccagact 51480 aattaaatca gaatatctga gagtggggcc tgggcattcc agtttttaaa atgcttagtt 51540 gtgtggaatt ttcgagctct aaaaacatac tttgttgaca tatcataaac tagtaatttt 51600 gcaaaaactt ttttcataag ttctcagatg tacatgaccc taattaataa acctcaggac 51660 tcagatttca ttttagtgtc ataagctttc aatttgttat atttgagttt gggattcatg 51720 agtgattatt gaagccaaca gagagctagg atgacagatt ttgggatgtc tctaaagtct 51780 ttccagccca gcttatccta ttaagaggct atattcaaaa ctcaacattc agacaaccag 51840 aacagaaggg cagatgagta ataattgagg aatctcagct atgtcaggat cccaaacacc 51900 aacctttgta ggagtagcag agctcaatat caagatatcc ttgtttggta tgaggattat 51960 aggaattgca ggcttattcc ttaaatatag ggggaatgga ttcacgatca agagctgcca 52020 ttaatttcca tagcatcttg gataacaaat aagaaaaatg tcaagagagg aaagcaaagc 52080 ttttgacagt ggcctcatta agtggaaaac atatttcctg tattaggatg gggattctcc 52140 aagggaggct taagtgagga gacctcttct tcaaattata ccaatgtgga aaggagttct 52200 gtttatttga ggctatagat ttgtattgct ctttggtttc ctgtgttttg gtacatttct 52260 gaggtggaag aattccagat ttcaactttc ctgaaccaag cattgagagc cttaatcagg 52320 tctaaagaat aggatgacag taccttaaag agctgtgaaa atgtttccgg atgttgagag 52380 aacacaaagg cttccaataa acttcaatag aaaacttcca ggaatctgtc ccagacctga 52440 tcccttagct tcaaccatct atattccttg ggttccagcc cttctcatct ttgactcttg 52500 aactacttga aaaaggtgtt tggttggagg gtgttgcttt tcacagaaat tcaatagtct 52560 tatagagaac agacatgcat gtatctatca ttgtaccaat agttaaccaa acatgagtca 52620 cttttccctc tctcccttca tagtaaatgc atgactgacc tagtttagga acactctcct 52680 ctggccaatg aacaatagct gtagttcttc atgtattttc tttctttgtt gaatttattg 52740 cttcatacac aaaaccctga acgctaggta ctgttgaact tttgtgccat caaagagagg 52800 tctattgact cttcatcaag gcataatatt tcataaatag ggtgtttaac aagctcttcc 52860 taaatgtttg ccttctgcgg ccataaagac agtagctgta aatgtataaa gtaaatacta 52920 tatgttttat acatattgtc atccaattat acaactctgc caccaatttg aagataaggg 52980 gcctgaagct aagtgtgtat gtgagttgcc tgctgagtaa caaaccagcc caaaacttag 53040 tggttttaaa caataacaac atttattttg ctcaaaaacc tgcatttggc caggacttgg 53100 ttagaatggc taattttact tcactagatg tcatttggga caattcaaaa gctgggggct 53160 ggaataatct gaaatctcat tcattctctt gtctggttgt tgaggcaatc tgtcatctga 53220 aaacgttgct gagactatta gctggaacac tacagagaac ctcttcatgt gctctgggct 53280 tcctcacaac atggtggcta ggttccaggg gcaagcatcc caaaagagac agagagagag 53340 acacaggcag aagctgtatc accccctatt acctaatctc agaagtcaca cattgtgtca 53400 cttctgccat attccattca tccaggcagt cacaaaagcc tgccctattt taagaagaag 53460 ggaaatgtgg actccacctc tagctggggc tgtggaaagg ttatgaagga catacgggat 53520 tggaaatagt gctgcagaca ttttttctgc tggttggtgt caggggagga atttgaaccc 53580 aggtttgcat gagtctatac aatgccctta atactgtacc acagtattct gtgaagctga 53640 ggactgaaat ctgcctctac tgcaatggaa tttagctgtt aaatattaca cctgggctag 53700 tgagcagaaa cagccaaact tggaggcaga gagtgtggac tttgggctct cccaaggcat 53760 ggtgaaggtt gataatatta acatatgaga accattcttc cttcccaaga ttggtggaat 53820 cacttggaag acataccaag gccatcagtc ttacatttct tgagtactta caggtttctg 53880 agagtagata aataaaacaa atcactgagt cacattcatg ctaatggagc atagcaacta 53940 gattattgga aggtcagatc atttagtttt gaaaaatagt aataacaagg catattgtct 54000 gacttataag aaaacctcaa gtatctagta actggcaaga tatttaattg aaatgaggca 54060 aattctagaa taacatatga cattgcagtg agataggcta ggaggtggaa ccaacaaggg 54120 agacttgagc caagcaagcc tttcaacatt ttcccttgtt ttctttcctt ctctgacttt 54180 cctttctact tttttttttt aagcttaagg taagtagcat gtttataggc ctagaagcca 54240 gagttattgc tgacttggga agnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60420 nnnnnnnnnn nnnnnnnnnn nnnntgagcc ccacctggtt cttctcctgt tgatgtttca 60480 gatggtcgtc acagatcacg atatttaacc catgagttta acaaacaatt tcttagggcc 60540 gcaaggctgt tgggatttga aggggaatag aagaagctga aagagggaca cccagacagt 60600 gaaagtaaat ggtagcatcc tcgcatattt ctattggtct ttgacaattt ttgtttacta 60660 aacccagaat catagtttaa tacaagatta ataagacata atgaaaatgt agggagccat 60720 gaaggatacc atgccctatg ctcctggcat agctgattgc acacggatca gaggatgtcc 60780 atacaattca acaccagccc tccgcagcac ctggggagga cctgttacct ggctggaccc 60840 tcaggctggc tccaggcatg cagctcagag cactggatct ctagccagac cacctgcgtt 60900 cgtcctggtt tcaccacgat gataacctta ggccagttat tttatctctc tgtgcctcca 60960 tttccacatc tgtaaaagct gaaaatatcc cacttcacag gcttctcatg agaaataaac 61020 aagttaataa catatgtaaa tcctttagaa caatgcttga caatatagat atgttattat 61080 taacattatt attatatgaa tttttcatgt agttgagaag tccactattt tgctagttcc 61140 tccttcaatt cccttatcct cccaccacca ccaccagagg aaaacagata acagtatatg 61200 gatctggctt ctgtttttgt ttttgctttt ttcttcagtt ctcctcttgt tacttccacc 61260 ataactggcc ctggggaaat tcttcctgtg tcgggatgtc ttaaaggacc gagaacctgg 61320 tacctgtcaa gtgcttcttg agaagttcct agggaacgta catcataaac tcaagttagc 61380 accaagctat tattaaattg atagccacct gtctgagcat gctttattgg taactttacc 61440 tccgtgggaa ggtgtagcgc ccacactgta ccctcctacc tttcccattt tatataacgg 61500 agtccgtgct ttctcaagac tcaggggtgg gtctgcttcc agcactgagg aaaagtgaga 61560 actgaatagg gagtggggct tttggtgtag ttaagaggga ggtaaaataa ctgtaatttc 61620 atagcaatta catacagcca acactgtcaa aagttccagt tttccattat aaacctgagc 61680 ccctgctggg agcaatttgc aaggtagaaa aaagcacaca atgaggttct aatgtatgaa 61740 agaaggcatc gctgcctccc catctgaatg ccatagcaaa acaaacctca agggagaaaa 61800 cagtgtgttg agcttcgaga acgcatttgt tttctccctt tcttttgaaa aaaaaaaaaa 61860 aaaaactcag taagccccca ttcctggata ttggtcggat tgtttacagc tttgtacgag 61920 tcttcagtac cctggcccac acaggccctt cctgtcagtg gaatgccctg ccaggccacc 61980 tgcaggggcc atggtttcca gggcagccaa catcagagtc tggagcaaat gggttatttc 62040 ttcctctaag tattatgttg ggggccaata aaccacccta tggaaatgaa ttctacagag 62100 accaattccc caaccttttt tgtttgttca taggtttgct tgaaattaat tctttatgaa 62160 attcagggat ctgcagccca ctcagaagtg cagtaaagat ggacttgctt ttcagaccta 62220 aaaaagccta ctgttttgag gaagggaaat gaaatgacag aagcacagaa cacccaataa 62280 taattctgtt atttttacat atgttcctgt cttatcccca agcccaccta cagaaggcca 62340 taagctcctt ggaggaattc aattttcaat cccctgggta attttctcca gtgtcttgca 62400 cataactgat agtcaatatg tatttgttga gtaaaagtgt aacaaatgaa tggattattg 62460 acagtgaagg ccaagtacta gccagaacta agtggtgtat tgattaaagc cttgagcttc 62520 ctgattagcc tccatatagg gagatgggat caaagtctga gatatttata ttcatgacat 62580 ccaaggactg aggggtgggg gagggtaaac tacagaggaa catattttgg ctaggtagcc 62640 gaaggaaagc atttctaatt gttagagcaa tcctcagaat gaactgcttt gagagatatt 62700 aatacaactg actggtcagt ggagatattc aagcagaaac tagatgatta cttgggggtg 62760 tagatgttgg cattccaata tcatttgtgt cagactaagt gatgttgcag gtctattttt 62820 accccaaggg ttctatgatt tcataaagtt atgattctgc ataatcatca agttcaattc 62880 agcaagtatt tattgagccc ttacatgctg ccaggcattg ccctgcgtgg acattaaggc 62940 aaaaacgaat aatatcaggc taccgccctt tgaagttgct gggacatggt tttagtaagg 63000 tctcctcaga cctgcatttg ggtgttacaa tcttatgttc cccttgctaa ttgcacctgc 63060 ccactcagga cactggttct cttctttcca ggctggaatc gatggagaaa gcatcggcaa 63120 ctgtcctttc tctcagcgcc tcttcatgat cctctggctg aaaggagtcg tgttcaatgt 63180 caccactgtg gatctgaaaa ggtaacgtac gttgcataaa tgaagagtat gtttagctgc 63240 atgaacagag aaccttccat gaaggagggc aaaagccttc cgtggctccc agcagccccc 63300 tgctcacacg tcannnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn naaattagag gggctaaacg 64800 ccatctgctt ggttcaaata ctatgctctt cataatacct cagttatgaa gtttgtttca 64860 tctttcaaat gagaccttca atccctcaag gacagactgg aatcgtgttt taagtttgtt 64920 tgtatcctcc atagaaacta gtttttcaca ggtcctcaaa tactatctgt caaataaatg 64980 aacttgcatc cactatgcaa ttaatgtctg tgataactaa agagagcagt agacttatga 65040 aaaagcagct tccttcagtg cagaattgga ggtgtttgga ttaccatgtg tgagagagga 65100 cttgtatgtt tgctggagaa gagagtggga gagggaacca cccctgcccg cgtgcttcag 65160 atggtttggt tcctccttca catgatcaca cctggtccag ccaacacccc tttgacgtag 65220 accaggcatg tagcaccact ccatttttac agataaggaa actttcttac tgccccctct 65280 acccatgaac acacacatta atttctaaga aaactgattt attttcctgg cactacactt 65340 tagtgactga ataatcttga acttttcaga gcctcagttt cctcatctgt acaatggaga 65400 cagtgtcttc ttaatcaata ggattattgt gagaatttat cgaggcaggc catgtaaaac 65460 atttgcaatt ggtcctgtac ctgactcata gcaagaactc gttgcatgtt ggttattctt 65520 agaattatca ctgggttcat ggagctggca atccagtaag gaaaagaaga ccgatcctca 65580 ttacataatt cataaatcat acctggcaat acgtcatcca atgctaatga tatagtctca 65640 actataaata ctgaagcagg gtcaacaaag acagagggct gcaactctag ctaagcgtcg 65700 gtggccatag gcagtacttc ccaagggcgt ctgactccag tggccacaat tctccagaat 65760 cccaggaaaa aacccaccac caccaccatc accaccacca cacattcctg tatctgtaag 65820 ccacagagag gtgactgaga gcacacacgt tgctgccttg tgtcagctct aaaaaacagg 65880 agcaaataaa gaatgttctt tcattgtcta aaatgaggga catgcacaaa gtaaacaaag 65940 ggactaagta aattagattt taaaaatact ttccgtctca gttgtctgag ttccaagtgc 66000 ctcagaatgc agctggagag tgtgtgtgtt tggggaggga catgggagta gcacagtaat 66060 tgaaattctt ctcttcccct cccaaactga ttaccccagg aatttgtttc attacagact 66120 cctctactcc cactgtttac tttttttaca gtttctgcgt ttaatgtttt ccactcccga 66180 ctcccacaag aaatgtggca ttctcagaga gagcatttgg ggtgaatccc agcaccgtct 66240 tggaagtcgc caccattttc cctcctcttc cctaaacatt tttagtgtaa ccctcccctt 66300 tcccattttc cccaatctgc caaaccttct tccccctcat tttgctccat tcttgctctt 66360 gcattctgac cagctggcgc agacacagaa gcacactcct ggatattaac aggacctagc 66420 cgtgtcaaac acaaggccac tttcctttcc actgcacttg gccttcctgc aggtagtcag 66480 tgtgctcccc agtgcacgcc agaaatgcca cttagcactg ctgtgggcta agtgcaggaa 66540 gttcaattaa ttgttcagtt aattaattgt tcattcattc aattgggact gcgttccaga 66600 atttcaccca cttctccatc atctaggcct ttctttcagg ataatttagg actcttgggt 66660 ctcctctttc ccagggggga gaaatgcact gttgctttgg ttatcttgga tttctttaag 66720 tgtctgaccc agaaactaac aggcacacca aagagctttc tgtcttctta gggagcaaat 66780 ttgctgccta ttacatatat atgtatgtgt tacctaaaag atcctactgg gcccagcaaa 66840 gtttgctata agaagaaata actttcataa atcaagtgat ctatttgaat cccatcacaa 66900 agcagaagtt gcatttactt agaaaataaa ttgtttttaa cttaagataa catttaagta 66960 accccataat ttttaaaaat aggcacaatt tctgactcct aggagatttt tgatttatgt 67020 atgttgaaca gaatttttaa aatttaaaga aaaataactt ctttctagta aatctagtaa 67080 atgttctcaa aataagtctt attttgagaa cagtagatgc ttgaattaag ttaaaatact 67140 gtgaaaggta gactatggtg gagcatgaat actgtagcag tggagttgcc tactggttct 67200 ccccacattt aactgaaagc cgaaagcctt cataactgga ttgaagtttt taccaatttg 67260 agaccaacat ttgatttcta cttttgcagc taaaatcact catttttaaa tttaaaatgt 67320 atgagttttt attgctgttg ttgttttgag accgagtctc actctgtccc ccagactgga 67380 gggcagtggt gccatcaacg gctcacaact gcctcgaact cctgggctca agcaatcttc 67440 ctgcttcagt cttccaagta gctgggactg caagcacatg cctccatgcc cagcaaattt 67500 ttgtttgttt tttttttttg taaagatggg ggtcttgcta tgttgcccag gctggtctcg 67560 aactcctggg ctcaagtgat cctcccacct caccctccca aagtggtggg attacaggca 67620 tgagccactg tagctgaatc aaaaatgcat gctttattca tgtagccatt aaaaatatgt 67680 atctactgag ggcctcctat atgccaggca tggttctggg tcctttccct catggagctt 67740 gaatcgtaag gctttctctc aggtatcttc ttactcacca gtaggaggtt cttggttcag 67800 aatccttggt aatagcctac ctggtgtttg tggaggaata atcttagcaa taagaagact 67860 tttgaaaaca tcttcattta atagttttgt tttcaatcta atagtttgtt tattttcctg 67920 gctacccaaa gaccatatca ggggctgttt ataactcaaa aagagtattc tgttgagtgt 67980 gacaaaaggc tgaccaatgt tagacattag ttattagata ttaaaatata agttacacac 68040 aatagaagca tctaacttga acaaatggcc caaactccca attttttgta tctggagata 68100 tttattagga gaaaagtatg atctaattca ttaattttta aagtatggaa ttatgtgtag 68160 cgcatttttg ttaggcagat tggtgaaaga gcttttaaaa gcaaaacccc acctctcccc 68220 tgggtaatta aagcttggat ctgacttaga tagtcacctg gattctctcc attttttcaa 68280 ttctttcatt ttttcaactc cttcatcttg acttgttcct ctctcactga ctccactgga 68340 cacacttcct gggtcacacc acattctttc agaactttct ccaacctccc ctgccctata 68400 ctgcatacca taaatctgtt tcacctgaca ggcatgttgg atagcacagg gcatctaagt 68460 tacaatcaat cttaggtttc ccagaacagg aagcaccctg ctgtaagcca acacctgaat 68520 cttgctctct ctacagaaag ccagctgacc tgcacaacct agcccccggc acgcacccgc 68580 ccttcctgac cttcaacggg gacgtgaaga cagacgtcaa taagatcgag gagttcctgg 68640 aggagacctt gacccctgaa aagtaaggat ctgtttcttt ctggggctgt tgaatgggaa 68700 gggttcatgc tggatctata aagtcctggt gttaacttcg ggcagatgca tttccaaagg 68760 aacctcacca gtccagagaa caggaccctt tcttcaaaga gagagcctag gacccccaaa 68820 gggctgccaa tctcaaaacc actccacaaa atccactcaa gagtagatat ggcctatgaa 68880 caagagccag tcactcctac ctacacagag tgattttagg agccctagga taccaggcct 68940 ccctcgcccc agtatgttcc ttctctccag ctcactttgc ttggaagacg ttgaagtgag 69000 gacaaagttt ccttcatttc agaaccagag ccgaaagcct gacagaatga aaataagtca 69060 ggaaagggta acccagtgac ttaaaacaac atccagggtc ttaaacctga ccaaatctgg 69120 tctgagtcca caattaaaca gatgaaaaaa atcaactaaa agcagcagct cctcttctgc 69180 aaacaggttc agggagagta tagaccatag agctataagt tcttgttggg tgacacattg 69240 ctactgatgg aaaagagcag taaaaagaag acagatggct tctaaaattt acccaagcta 69300 gacttgttca ttaaagaata ctattatcaa ggattaaaac aatcacaatt tttgaaaaaa 69360 ctagaaaaat aatattacat atagagtgag gcatgaactt tatcctgtag ggtattatcc 69420 tgtagtgagt cctgtgggat attctgaaag ggattaagca caggaggtgg ggagtccgtt 69480 ttaagaccta ctttagaggt tgagggaagt cggtatccta gaggaccatg aagctggaga 69540 tagaggaatt aattctgggg agccattggt gactgtccag gcaggacgaa tgaagggcta 69600 agccaagaca gggcaggaaa atcgggggag aggagtagga agggttcagg acatactgag 69660 taagtaaagt tagtgagatt tgactactaa aaaccaccat cctgcattta ttttaaagtt 69720 ttatttattc atcaacactt ttatagcctt cattttgtgt caggcaccat tcacaacact 69780 tagctaatat gattcatttc atccttctag catcctaggt actatcatta tcctcatttc 69840 acagtcagga cagggagaga gtaagtcact tacccaaggt tgcacatcta gtgaattgct 69900 gttaaaaaat taaaactgcc agtcatgctc caaataagcc ttaaacaatc atatattctt 69960 ccttaaaaat attctttgtc atccatattc agtcagctta tttgcacact gagagtgaaa 70020 aagccacctc aagcccattg gaagcagctg agtatagact ctaaataagt gcattaatta 70080 aatcaatagg acagccccac atacacacac acatctctga agaggacacc agccagccac 70140 ttcactggaa gtgccctgag ggagcccaga ctccatcccc ctccccggga ctcaacctca 70200 ttgctagcac tgatttgaaa tcccacatga aaaaaaatgc tcttttcaat aactagagtt 70260 tagactttgc ctgggagacc catagaggaa agttcatgac gcagttaaca ttttccctct 70320 tagaagtaac actggcactc aggagtgggg gcttccgtca tgatcattag gaagtggaaa 70380 gtggaagcag tgaggagcag aggggcccat ttggtctgtt gccaggcagt ggatggaggg 70440 gtgaggggct ggggtacaat cctagtctgc tagtgatgtg ctgtgacctc ttgagccagt 70500 cccttcacct cttgaaccag tctcttctct gctctaggac tcagttccct tctctgtgac 70560 aggatatctc ccagagccct tcttgctctc tgattcttca catgcccttt agccaggttc 70620 cctcaccact accacaccac ccccagtgca actgtgcctt tcctgggctg cttctctaac 70680 tcaggggtct ccaatctgca accacggtgg gaccacaagg tgggcagaga tcactggaat 70740 tgtatgcccc atgcgtcctt gaacattctt ccagaggcga gtcatcatct gacttcgtca 70800 tcatttcatc agattctaag tttccatgat ccaaatagag ctgggcaggg ctggatgtgg 70860 accctccctc cactccctcc tctccaccta ccttccctgt cctgagctct gctcccgctg 70920 gtccaggtcc ttctctggac tgcagttgtg tataggaggt tttatgaagt gcattcctgc 70980 catgttagtg acctaacaat ccactactct caggtagcag aacgcttgat gctgttaaaa 71040 tgcatatctc cagaagccta ccaaggtggg ggcgccaagg gtgaggccat caagtattcc 71100 agagcatccc aggattatac cttagatgcc tttgcaccca tcagactgag gcaggtgcca 71160 caattacctc ccagtaaatg tcaccaagcc agtgcctcag ccccatagag acctgctggt 71220 ctgcccagaa caaaccacca gcctcaccag cttccccagc gactcaggct gggagatgga 71280 gtggctggga ggttgtcagg cctctggagc aggacagcct ggccttctgg ttctctaggg 71340 ggcagcaaaa ccctcataac cattcaccaa gcgctggtcg gccatgtgtt tctcatcata 71400 ccactatttg taaaagctgc caaaagccaa cacattatgg gaggcttttt cccttagctt 71460 tattttttaa aggagaaaac cacatacaaa cactccaacc atttttttca aatcacattt 71520 gatcccaggg agcatttggt gtcaaaatga ggaatccagg aagtatcaag ttgtttctaa 71580 atgagactaa catccatccc tctccttaga ttgatctagt gaacacacga caaggaagtg 71640 ttgtgattaa tgctgggatt actgttgaga cattctggcc cttgcctttc atctcctgaa 71700 gtggccaaag tttctaggat tcagcactga gagtcaggag ccctgcctct ggccacctct 71760 ctatgagtgt ttgtcatatg accttggaca agcctctctc ccgctcagac cctccttgcc 71820 ttctcatctc taaaatgagg gggtgaaact acatagtctc caagatgtct tttgttgcat 71880 gttccacaat tccatgagtt tctcaaagta ctcttccaga gctggttatt gtctcaaata 71940 atcctagcac atagatgctg taataaaaat tcaggagggt aaactgatac agtctattta 72000 cacagcatgt catttagtca gtcatctagc aagaatgatt taagtatcaa cagggttcag 72060 aacactgaca gaattggaag acaacttgaa ttcattattt tacaaaatgt atagcgataa 72120 ctcaccacag taaaaaacaa gaaagttctg accctctggg aggtagagaa acagcctcca 72180 gacctcccac aggatggcat ggtagctggg ggcaggaggt ttggagtcag agtgactcgg 72240 gttcaaatcc ataccactag gttgtgctat gttgcagtaa caaagcggcc ataaaattcc 72300 agttgcttaa cagaacacag gtttatttct cacttgagca aattctgaga gggtagggtg 72360 gccctcttcc atcctgtagt ttttggaata catgccctcc aaggctcctt cagggagaga 72420 gagaagaaag atctcccaaa gtgttttgca gggccaggcc tgcaaaaaaa atgtcatggt 72480 gacattactt tccttcctat gccattggtc agaatccaca cgtgatccca acttaggtga 72540 gagagaagct gggaaacaga attacatggc tatttagtga gtatgcatat tctccgctac 72600 aggcaaatga cctgaattct ctagaatctt taattctcac atctttaaaa caagaaccat 72660 actgttgatc ttatagggct attgtaaaaa ataatactaa tgagcctaga gcctgatatg 72720 aagtaagagc tcattaaatg gaagctcaag gagttagaaa gagtggccag tgcctgtttt 72780 gggtctgtct cccaggtgca attgtacaga caattggtga ccttaggtct taaatcccca 72840 gatcctaggg gatatctttt aatgaataag ccaggcctct tccacctcca gagatctttt 72900 gcttacacag cagacatgag tcaaacaagc acacagtcaa ggattagaaa agtcagatgc 72960 attaacgtcc ttcacgtgtg aggggttgat gaagtaagga agtgggaaca atggagaagg 73020 gagagagaga ggaagtcttt ccacttttca gctcgtgaga aaaaggaggg aaaacactta 73080 agctgccatc agcaaggcca ttccatcagg accgtctttc tttttctcca gggtatctat 73140 ccccaaaacc aatctcaggg cctagtcctc agtaggtgtt tcatgcacat tggtttaatt 73200 gcatggtggg tatccttttg ttcagtgtct tggttggctg tggctagtca gtgagaagtc 73260 agctgggact ctaaaccaga tagggcactt accatctgca atggttttct ctttttttgc 73320 tactattttc tacttttgaa aaattttttt tttttttttt ttttttttgg tgagaagcca 73380 actgttttac ttcccagaat gaaagttaag tgcatgcatt tgaaataaga tatcctaaaa 73440 accgggtgga gagaaatttt catcagttag atcaggagtt agcagcttta tctgtaaaag 73500 gtcagatagt catgtatttt tggctttgtg ggcccttcag tgtctgtcac aattccacag 73560 tgacactgtg aaagaagcca taaactagta tgtaaatgaa tgggcattgc tgtttcaata 73620 atattttact tatgaacact gaaatttgaa tttcatatta ttttcacgta tcacaaaaca 73680 gtatcctcct tttgattttt ttcaagcatt taaaaatgtg aaaaccattg ttagcttgag 73740 aacctcagac aaataggggt catgggtcac agtttgctga ctcctgagtt agaaaaaatt 73800 atcatctctt tgattatcat cagatcacat ccaatgttat ttcaaagctg tttctgtgag 73860 tgaaacactc ttccttagcc ttttagagaa acagaatgta ctttgtagcc cgcttatttg 73920 catatttaaa acacgccata aatatggagg taaaatgaat gcacttatcc tatatagtat 73980 cctcccccag tgtagttact gaaagttgcg agattaacat cttagagacg cacttcccac 74040 aagaacaaag ctcaaatgga acactgcact acacagtaaa atcacaacga gtcctagtgc 74100 ttgggaaaca ggatgacacc accacagccc cctgaataca cgatcctctg tcatcatctt 74160 ggatttggta ccacacatcc ctgtttggcc caacctgatt gtgaagaaat gatcagatga 74220 ctgaaagtct gaccaattga tgagatacag caaaacaaag caagaaaccc aaacaagctg 74280 tccactgagg tgcattttct aggcctttct gtctttacat tatcaaggag aaacaaagaa 74340 cacttggcat ttgttaatag caatttaagt ctatcgattc taatagcatg ctggattcta 74400 tgtgagaagg ccaaaataga aagagatgac actgaattcc tgtgtcttgg tagattacct 74460 agtatcttgc aggaagccct gctgtgtgcc cccagccaca gcagatgaat gtggtgattc 74520 tcagtatacc caggaagaca tgtgtgtgga gcatcggtgc aaagcacccc ctcctgatgg 74580 atggagacag ctcctccaag gtgggcagct cagcctttaa ggttctgggg ctttgagaat 74640 caacagcgga gtttatgaga agccacacaa tgtggaattt aaaacctagt cttccacagt 74700 gaaactgcct gggttgggat cctggctctg ccaggcagcc atgtgacctc aggcaaattg 74760 cttaacctct ctatgccacc gtttcctcat gtaggaaatt aggctaataa tagtaccaat 74820 agagttgttc tgaggagtaa atggtgaata cgtgtaaagg gaatagaaca gtgcctgcct 74880 gctgcacagt aggtgctcaa tgcaaattac ctttcactgt ttccagagaa gcagtggagg 74940 gggaaggaca ctgctgtagg gccaggaaac caggttctca tcgtccctcc accatttatt 75000 agccatcaac atttatcttc tcaggtatcc agttcccttc catgtaaaag gaggacaata 75060 atgccttcct tatctgcctc acagcagaga gctggactct tccaatggca gtatctctga 75120 aattacacag ccacctaaca tggccccaaa aggggcccca aaaggaatcc tagctcatgg 75180 gccagttgag taacaaaaaa gcattaaaga acaacaaata ttttttaaag tcttgtttgg 75240 ttcgaactat caaaaacatc aaaacaaaca aaaaggattt tgctttttaa acgtaaggca 75300 acccaccagc ccacagtcaa aaacgattca cccagagatc acagaggtgc acattcattg 75360 aaaataatga tagcctggcg actgtctccc atctcttccc accactacct ggccataccc 75420 ataaaatgct tatgaaataa tatttatttg agttgatttt tttcattttg atggggctaa 75480 tgagtccaag ccctgggttc ccacatgagc aagtttacct ccttcagtct caaggccaat 75540 cgttctaact ccagcctgcc tcttgtatta ttgtggcttc aaaatgaaag actctacgaa 75600 agagagagag aggggggctg gatcattgca aatgtatctc caaataaaat aaaataaaac 75660 aattgaaaac acatgtcctt ttttggagct tggtagtttc tgcatgttat tactgcacac 75720 aaataataag catgtaaggg catttcagaa gaggaccgtg ggtattcttc ctttggagaa 75780 atggtttaat gctaacatac ctggcttctc cttgctccca gaattagctc tgtgtagcta 75840 ggattataat cacaggactg gcagtagatg gttattttag cgggtaaggg aagtgttgag 75900 aaccctacca gatagagagg caagctcaga gggaactttg attgcgccat cccactggca 75960 catccttatt ggtggtccca agaaaaggac acatcctgag acccctgaat ggcattctgg 76020 ttctttgcct agcagactgt gtcaccctag gcagatctca gcacctcact ggcctctggt 76080 ttcttctatg gatgccctag ggctgagaat gcccaccctc cctatagtat gaggataaag 76140 acccccaaaa cattttgtag atgcagctgg aaagggaagt attgcttatc atgataggga 76200 taggctcacc cttaaaggag ctccaagtct actgattcac agaacactgc atcctccatt 76260 ccaggtaccc caaactggct gcaaaacacc gggaatccaa cacagcgggc atcgacatct 76320 tttccaagtt ttctgcctac atcaaaaata ccaagcagca gaacaatgct ggtgagtgac 76380 tcccttccac caagaacctt tggcagtggt ctatctgctg cccaaggcat cttcaatttg 76440 cccataggca tgtcaaatac ttgggccaga gaagcatctg gaaccaatac tgcttattat 76500 ttgcccccac ccccccgcca ccaccatctt gtcccccttg acacagggga ggaggtggtg 76560 gatgctgctg cctagctggt gcaggaggaa aggcttgggg ctttactgcc aagctggaga 76620 gagggatgct ctttgtcatc tgagcctgcc ttattgcaca gcatagtgca ctgcacggaa 76680 cggtcctcac tgcccatttg tcccaagttc ccaaccctgc ctgtgatcac attagagcaa 76740 catattcccg cttccttctg gtccctacaa ggtttctcca gggatttttc cagaggccaa 76800 gggagaggca gcacagtcac tactgtggca aatgtggaag gcagacctgg gctgtgttct 76860 ccccttctgc aggacagtgg gccctggaaa gccactcctc aaagcctggc ccatcctaca 76920 tcaggccctt catttctgac attggctaca gaattcagga cttggttctc ttgcggggcc 76980 ttctgaaagg gagctgagtc ccccccagtt atgctcacag cactttcagt ccttcatttg 77040 gcattgccct ttaagaatta cctaaagcca tttgaatgga accgtccaat taagcaggta 77100 taacacttga agaacggtgt taggcccagg aggaacaggc tagccactgc agtgccaacc 77160 tggggggctt ggatttttat cctatgggca atggcagcca ttcccttgcc ctcagaatgg 77220 gatgacagga cagcctcaca ggctggatga ctggagtcca gcacaaagcc aagcaactta 77280 agtgctccat gaatctttgt gaatgaacaa gtgactgatg cacaatggac caaaacaagg 77340 gaatagatgc tagatgcccc agaagctcca aggagaatag catctctttg gatcaaggcg 77400 accagcatat gcagctatgc atggtaaaag agacgaaatc atggcaggaa cagagttggt 77460 gtatcgggaa gtaaataatt caggaagcta ctcaggaccc agattgtgac aagtgcaaaa 77520 cttggttctg gaggtccaaa gaagaaacaa acacttcctc agtcttcaat gcctggcaga 77580 tgcagaagga caggcttcaa gtcagtttga tccaagaggg tctggagagc tgattaactg 77640 caggtaaaag aacaaaataa tttctgtcga aggtattcag aaccagaagc atttaccatg 77700 ggggtaaatg catgagactc cttttctggg agtttcaact gggtcataaa gattaagaag 77760 gaagcagttg gctgagatca ttgagcagaa gatgcgggga agttgagaaa aagtcaggga 77820 cagagactgg aaaaagtgac tgaagcccac agtgccagct tggagggctt gggtttttat 77880 cttgtgggca atgacatcca ttagaggtgt gtgtcaagaa actagaaaga gagtggcttc 77940 tgggaaataa gaagaagcat gtggaaagta ctttcaatct acttggtgct cttgcttcat 78000 tgctgcttga accctatagt gtgttgagta gtgtcccccc agaattcatg tctagcctgg 78060 gcaacatagt aagaccctgt ctttacaaaa aaaaaaaaaa cttgttttta attagctgag 78120 caaggtgatg tatgcctgta gtcccagcta ttcaggaagc catgacaaga ggattgcttg 78180 aacctgggag gtcgaagctg cagtgagcta tgattatgcc actgtactcc aacctgggtg 78240 aaagagcaag gtcctgtctc taaaaaaatt tttttaaatg tatgtccacc tgggatctca 78300 gaacgtgacc tcatttggaa atagggtctt tgcagctgta attagttaag gatttggcga 78360 tgagatcatc ttgcatttag tgtagtccct aaatccaatg actgttgttc ttataagaat 78420 aggagagggg ccaggtgtgg tggctcttga ctacatccca gcactgtagg aggccaaggt 78480 gggtggatca cctgaggtca ggagtctgag acccccgtct ccactaaaaa tacaaaaatt 78540 agccagatgt gatggcacat gcctgtagtc ccagcttctt gggaggctga ggcaggagaa 78600 tgacttgaac ccaggaggcg aaggttgcaa tgagccgaga tcacactgtt gcctgggcaa 78660 cagagcgaca ctccaactca aaaaaaaaaa aaaaaaaaaa aagaggagag gagaggacac 78720 agacatgtag aggagagaag gccatgtgaa gacagtcaga gactggggtg atgctgtcac 78780 aagccaggga actcttagct ttccggaagc tggaagaggc aaggaaggac acctactaat 78840 accttgatat tggaactgta aggggaaaag tctctgttgt gttaagccac ccagcttgtg 78900 gtactttgtt gtaacagtcc taggaaatga atatggcccc tcagagccat tgtgggaaag 78960 aagagtcatt gctgctttcc cggctgagga cagagaacca aacgagaaga cccatgactt 79020 gtctaccccc aaggcagagc tggctctcga accctcgttt tcagactcca cacccagtat 79080 cctttcctct tagccacaaa tatcttcttt catacttgtc aaagtgccct cagaaagatt 79140 atctcatttg agccctccag atttttaaaa ttataaatgg tgtttacata ttatccatat 79200 ttacagttct cctattccca agtccaaagc cagtttccac caaatgccgc atatccattt 79260 cttataaact cagtactctc caattttcat tgtttcccct tcttctgttt caccctgttt 79320 tcttctgtaa tgagacttgc ccttgtgact cctgcttccc ctaactcatt tgcacatttc 79380 aaagctccaa cattgctgct ttcccaaaga gcctgtcccc agatgtgcta ttccaccttc 79440 tgtttaacaa gacaccacac attccttccc tggaactata acttgcccac acccctccct 79500 tcctctttac ttgtccttcc tcccacccaa tacccccgag ttgtggtcaa ctgggtccaa 79560 aattcttagg cttagcaagc cagaggtgaa ggctcctgta aggcttgccc tcctgtagag 79620 agcagcatgg ctcctgtgtt acggtcccag agccccacag cccctgaccc gggagatggt 79680 ccatcccctt tctgctctgt gtgagctcca tggctgtggc ctctccccaa gagcatcagc 79740 attcaggccc aggcctcctt ccacatgccg ccctacatta ctttgccatt tttttccctc 79800 tggatcttca gctctgcacc tagttctcca actttctttc tttagagggt ttctcttgta 79860 ttttcattta ctttctcttg agcagagccc tccctcccct ctacctggtc actaatgccc 79920 cagcccccat acacacactg gcccaggtca tgatcctttt ctgccctctc aaaatgaaag 79980 catgagaaaa gccaacactg ttacatggtt ctccatggtg ggtcacctct ccactccact 80040 cctgtggtgg ctacaggaac caccaagtaa aatttccctg ttgttcccca aagccttgta 80100 agtccccagc taccacaacc gagaccccat gccgtgggag ccatcccagc agtactggag 80160 taatggagag aatcatccat tagtttgtac attcagtcat tcactgattc attcaaaaaa 80220 ccacaagtta tataaagtga ctacaagtcc gagctaatat ttaaagggca ctgcttatgt 80280 gccaggtact gtgctaggtt ctggagttgc caaggcaacg agacaaagtc attgccctcc 80340 atgaactaac aatctacagg gagctgtaaa acaagtacat gtgcaattcc cggcctgggt 80400 ttgaatccca gcacttctag ctgcacaact ttacccaaga ggcttattct ttctgagcct 80460 ctggctctgc atctgtaaaa tggactttac attcagtttc tttatctggc aataaggtaa 80520 ttaataatat caccaaggtt tgtaatgcag gttcatctat tttataactc agcgcatgta 80580 aaacccttag cgcagtgtct ggcctcgtaa ctgttaaatt tgattattct gattattgct 80640 attagcacaa aaatgggact aagatgaata atgacatcag aaactgctgc ttcagctctt 80700 gcttccccct cagttagctc ttccaaatct cccannnnnn nnnnnnnnnn nnnnnnnnnn 80760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81360 nnnnnnnnnn nnnnnnnnnn tagacaattg taccttagca gagagttctg gtataaaaat 81420 gcagtttgag gggttataca tctttagatg attaagacga tgcccaagga tcaatcacat 81480 agatgggatg acagagggag aggagtcaag gaggaagaga aagaaggact agccagggag 81540 gatggaggaa atcgggggta gtggggaggg cataaagatg ggaaagcaag ctttattgtt 81600 gttgttgttg ttttaaaaga gtgatctgtg ttataaaatg ctgcagagaa ggtcctggga 81660 gacttttgcc tgctatgtgc agctccctga aagtgttctt ggatatcctt ttctgtctgc 81720 ttttctcatg tggaatccac ccattactca gattctacac aaccagagag agctcagtct 81780 gatggaggaa gcacaaaccc tgctccctta gaaaggggag gaccagtggg tgatgatagg 81840 gaactggatc aggaacccac atgggaagag tgctcgtggt cctgcgaaag agcatctttg 81900 agatgggaga aagcagcact gcatttagat ttggctggag gggttcctcc tctgagcccc 81960 acattcactt tttcaatctt gcagggagaa gttatgccct gggtttagac ctggaggttc 82020 atagagtgct gaagtagaag gcttgaggga gggcacagtg tcatccatgc ccctttggca 82080 catcctgcag catccctgta attagcaagc agttaggtct gtcctccccc ttaatcatgc 82140 tctctaccca gcctgcctgg cacatggtgg agttcagcaa gggcccaggc ctccaccttc 82200 tgcaagccag tgcctccacc tctagcaagc gtcgctgtga aacacccctc cccgcctgcc 82260 actggaacac ccctttggct ccttgggttt gtctcttccc taaaggggtt tctttgacac 82320 ctactttaac atgtccccca ctctctgtca ttcctaagag gatgtggact tctccctctg 82380 aatatctgtc ttacattttg catttcttac cctcaatctg ccatttgaac agactctaaa 82440 aatccctgca aatctcttgg ttttcaagaa attcatttta gttacttatc ataatctagg 82500 cagaaagtgc tatatttcac tttattttcc acaatctctc tttccttatg cttcaatcct 82560 agcaatacac atgatttann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 83040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 83100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnncctcc ttttgcgcag 83160 cccacttcca caggaactcc ctttccccta gtgagaggtc tgaaaagccc caggggcaat 83220 agtatctcat ctgtatcaca tgcagtcttg gggcttaagc tctctcaggc ctctcaggcg 83280 acctgcacta agtggtgttt tgcccaatct ggatggtttt agtctttcaa ggcttccttt 83340 gaattccctc ttttcttccc ccttcaagtc cacttttctc tcttatttcc acctctgtat 83400 tcctctgtct tggggggcct tctttctatt ttaaaattta tactccactt gttccagaaa 83460 ggctttatag ctactcccag ggatgcataa agcacggcaa accaacaaag gccaagagga 83520 ggtgagaaag aaaaacaagg agacaaccat caaatggagc cctgggtgag ctaaaacagt 83580 cagggcagcc ccggggcctt cgtgcgccac acagaaggta gaccacaaat ttggctctaa 83640 acttgtgagc agacaaagca aaacagggcc cctggtcagc tgcagctcgg tcctcctaga 83700 agataaaaac aaaccagcct cccagaagaa taacaaatat tcctgctgtg agccgcaggc 83760 acggaggagc tgtggtttcc attttctttg gctctcccct cctgtgactt ctctttcagg 83820 ctgctgtaga ctccaggccc tgctgacttc ctgctgctct gactcctctc catcttcccc 83880 cacctgtgag ctgtgctcct cccgatctca caatccctgt gttctctctt cacctgaaac 83940 tcactttctt cctttctctc catcttctct ggaagactca ggagacctcc ctgcagcctc 84000 aggcgtgtgt cgccaacctc cctgcccctc tctgtccacc cagcacatgg gacacagaac 84060 agcctgtctg ggcttatcta ttttcatcaa gatgaatggg ctcctgcact agatgtgcaa 84120 agatgtaccc ctttcactcg caaggctgca cctagttctc tttttatgga agactctgag 84180 atgtcctaac atactcttcc taaaatactt gtcctaataa ggtgcttcta acttccgaaa 84240 gaaagcccta aaaaccttcc ttggtatctg tgacgtgctg ggcatgtgct cagattgcat 84300 tgcccttcat cagttgtaac tgcagtggcc atgttttcca aactgaaaat cagatgccta 84360 acaaattgcc ttcattgaaa ataatcaaat tataagaaat tggtactgtc ccaggaaatc 84420 caggacacat gtccccagat gaaaagcacc tggttagggc taagccctct gcttctcctt 84480 tctctccaat ccaagctcaa acgtggttac cagagggcga gggagtgtac cctagttccc 84540 accacctcct ggttcccaat ttgttgctgg aacaaaaaag tggccattag ccacacaggt 84600 ccctgagcag tcttggttct gtttccaaat aaaggccaag gtttccacct ccctaaggct 84660 cacatcagag gctcctcttt ctggaacctg gaagctccct gggcttattc tggaaaaatt 84720 gattgtccct gccacataga atggaagaag actctcgggt tattctagga acctcagtga 84780 gtttatctca gaatttggat gatcattccc actgaatggg aacgtgattg aaggaggaca 84840 ttgtggttct tattaacaca atatcaatga ctgtcatcct gcctctctca tttcccttcc 84900 tcatctccta gagccctcat ataaggcagc aggggcaagc cagccacagg tgcattcctc 84960 gctctttcct tgtcttcttg ttccattcag gcccgaatct tcaaattgct cctgtcttct 85020 ccttctaaga ggtgccacct gcagtgggtc atgctcctgg ctgatgctgc tagaaaaaga 85080 actcatctgt atcactggcg gtctttcttt tccttccagg ttttcatggt ttgtgtcccc 85140 aacaactcat ccctgtcctc ccatcttgca gactgcctgg tgtgaccaca ccgacagact 85200 gattcctgca gccggctctg gtcctcccta ccccgggggt cacagtgctt ccttcccttc 85260 gctccctttt aataagatct taagactctt aaagtaaacg tattaagcaa gatcatcctc 85320 atttgatgta tagaaaattt aggcttacac acttcagctc aacattagaa agtttacaat 85380 taggccgggc atggtggctc acgcctgtaa tcccagcact ttgggaggcc aaggcaggcc 85440 gatcacgagg tcacgagttt gagaccgtcc tggccaacat ggtgaaaccc cgtctctact 85500 aaaaatacaa aaattagctg gtcgtggggg catgtgccta tatcccatct acttgagagg 85560 ttgaggcagg agaattactt gaaccaggga gttgtaggtt gcagtgaacc gagatggtgc 85620 cactgcactc cagcctggcg acagaatgag actccgtcta aaaagaaaaa agaaaagaaa 85680 ctttaaaatt aaatctgtag aatggactgc cagcaaaaga agtgagtatc ccatcaacag 85740 aaaggaccaa aaagaggctg gatctttgga ggcaggaact gcctaatcta aggtgtcatt 85800 gattgtacga cactattatt ttaaatactg gctgggcaca gtggcacaca cctataatcc 85860 cagcactttg ggaggccgag gtgggcagat cacttgaggt caggagtttg agatcagcct 85920 ggccaaatgg caaaacccag tctctactaa aactacaaaa aaattagccg agcatggtgg 85980 cgcacacctg taattccagc tagttgggag gctgaggcag gagaaccact taaacccggg 86040 aggcagaggt cgcagtgagc caagatcatg ccactgcact ttagcctggg tgacagagca 86100 agattccatc tcaaaaacaa acaaacaaac aaacctctaa gaaacaacaa caaaatgcta 86160 tcacttaaac aaagtgtttt cacttaacct ctcatatttt gattgtaaga cattgcctga 86220 tttcacagat gttaaagagt gaaaaaaatg tgcaccttag acttgagaca taatagtgga 86280 ctttcacagt cctttccaga tctaaccttt tattacttcc ttgaggctat tcagtgacat 86340 gtcagaccct ccttttgctt gaccttgagc tgccctgttg gaaaaaaagt gaatcaagtt 86400 caacattgct tctaaattgc cctgggatag cgatactagc tccatttcca gtaaatagca 86460 actctgttct ctgtccctct tgcctggaaa caggtagctc ttcctcataa gtcagggctt 86520 ggcaaacttt ctctatatag ggtcagatag taaatagttt aggctttgca agctacatag 86580 cctctaccag actctgctca attctgctgc aggaatcata aagcagccac agataatatg 86640 taaacaaata aatagggctg tgttccaata aaacatcatt cgtggacact gaaatttgaa 86700 tttcacatca ttctcacttc atgaaatatt attcttcttt tgactttttt ccaaccatgc 86760 aaaaatgtaa aacacaaaac aatacttagc ttgagggcag catgaaagta ggcagtgaac 86820 tggatttagt ccttggactg taatttgctg acctcaatct taggcagtcc gtggctacaa 86880 tagcaagttc tgttaatttc agtatgcctc agcctcctca acggtaatat ggagattgta 86940 atagcaccct ctcttgaaga tttgtcatgt taattagatg aattaatgca tataaatcac 87000 ttaggacagt tcctggcaca cagtaggcac caatatgtga tagctattgc tgctgctgtt 87060 gctgctgctg ctattaattt cacagtgctc cccccgcctc catacctagt gtccaccctt 87120 tggactgggc agcccagctt tccctgtaat gaggaagcat gtgctgccct cttctggcag 87180 gcagaggaat cgtcgccaga gccagctgat gtgatttatt caaggcctgt ggattcgaaa 87240 gggtcttcct atacaaagat tcagttttat ctaagccaaa gcttagacgg ttcaaacaag 87300 gctaggtctt cttcaaggct caaagccacc ttccattatc tgtcccccac accaagggaa 87360 gggcatgtcc accaggcctg acggatctac cataggaggt ccctggctgc ccctcccaat 87420 gacaactacc cccaaatgct tctttctccc tttcctcttc tccttctaca acctcgagtc 87480 tttacattta cgaacttcct tgacctaaaa taaaaaagtg cttgaaagga ttcagagcgg 87540 cctgggaggg ctgttagcct agaactaaca agccccttat tatcaaaggc cctgtgcttt 87600 cccccaggcg tcatgaaagc cactgctgct ggggggtagg tggggaggag ggtgagggac 87660 agtgaagtgg ggcatttttt ccagattccc aggatgtggt gagccacgtt taaaaaagag 87720 tgtgtatatt tattattcca agccacttca atcataaggc gcttcatgcc ttctcccctt 87780 cctccctccc agcccccagc ccccgtgtca cagtggccct gtctggaggg cctggccatc 87840 caggggatgg atgcggcacc ccccttctca ttctgccttg tgctctgcca cgggcctgat 87900 gagaaagcta aggccgtgtg taaaaagcag caggaatgtg aaacctaatt accctgtagc 87960 atgctcagaa acagagtcac ttaggtggcc gtggggctcc atgtctactc tgttggaagc 88020 agttcttttg gaggacagtt gggtcccagg aagaccgctg cagaggcctg ggagccccac 88080 ggtgggctgt tatcaggcca tcttggtgaa ggctcctggg tggtcaggcc gacctccaca 88140 gcaatgcgga taacatgtga ccgtctcacc acttcctcat aaagcagcca ttgctgtctc 88200 ctttcaaaaa cgcctctggt cacaaaggga cccttcaggc tgccttcctt ggcaacccct 88260 gtgtgcaaat agccaactag gggtattttg tttgttggtt tgttgggtta aagttttgct 88320 ttgactagca ttcttgtaag gaaaaacaaa taacctaggc tcggtgggac ccacctcgga 88380 ggtctggtgg gctctgcact tgcatgtcct cgtctcctcc ctcatggagg gtccttgggg 88440 ctgagggcag gagggagagg agtgggcaaa tgcttcctct ggtccctccc atgagctgcc 88500 tgagggattc ctccagaggg tctttgattt ggttcattat tgaggtggat gcttccaatt 88560 tataatttaa caaatcttgc tcctgaaacg aatccttggg cagttgtaag gaaggcggtt 88620 ggtggtggcg tggcctgttg aatggggcag gcatggggag aagggggtgg ggagagctgg 88680 gccctgcgtg ggtgggcgca ttcattagtg gaattttgct gtgtgggctc ctggactgga 88740 gtagagcgga gaccaccaac accattccat gtggtggaat ttcccgctgt ctgaccagaa 88800 tgagttctct tggcctggcc tggatgcaga tcaagacctg gggaataaag gaacgttaaa 88860 taactgattt gcacatgagt taaatgttct ttcacctctt cctaccgtta cctcatccta 88920 tgcaaccaac acctcttgtc tttgtgcagt ctacacatag gaaccaaata cgtttctcaa 88980 tctctctctc tctccacaca cacacacaca cacacacaca cacacacact tatgcacttc 89040 gtgtgtgttc tgatgttctt atgtactggc aaaggccatc acatacctcc ttagacaggc 89100 tcatttcaat taatctaaaa gaaagtgctt acatgcaaaa ttatattcat gatgggacac 89160 aatatattca tgatgggaca caatatccca ctaagcacag gatatgcaga ccacagaagt 89220 acaatagtgt gtccatgggt gtgtgtgtct acatttcatt gtgtgtgtgt aggttgaaag 89280 tatcacagtt ggtttataca attactacat tcataacccc ttgtctgtgg ttaggacgta 89340 gctttgagga aactttttcc aagtttctgc tgacctttag gggagaatat ctctggataa 89400 gttaacctgt agaacagatt ccctcatttt tggtagagct ggggctgaaa atgacaagat 89460 gtgnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 89520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 89580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 89640 taatttgttg ggaaaagttg ctacatggta actttggctt taaaacccca ggctttcttt 89700 caaatcctgc tccagcgccg tcttgcagga aggcactcta ccccatctca agcttgtttc 89760 caggctgagc cggagtagcc tcatgagtta agacaggcac agacttaagg cttaagcata 89820 aagcagcact tcttttcatt cctcttgtta gccagaccat tttctacctt gcgaatacat 89880 ttgaatcccg tgggggtttt agaggaagaa taacccccat aggttcttag ttggaaggga 89940 cccctgtaac aaaagacaga ttaacaagag aaaaacaagc agaagtttat taacatgtat 90000 attttctata atgaggagtt ctcaaagaga tggctttgaa ttctagtttc acagcatcct 90060 catcaaagga cagtaaactt ttagagatgt gacaagggaa aaaacacttt gagtctctag 90120 gggcagcaac ttcgggggat agcaaatgaa tggcagataa aggccagtta gcaaagcttg 90180 ttcaatgtag attcctgcag ggccatctcc aggagcataa gcgtcttaag ttgttttcag 90240 tggttctccc tggtagaagg ggggaggcaa gatacgtttt gtctttgtaa atctacgtcc 90300 tgcttttaga caaatagagg gagggcagag agctttactg cctctgtttc ttctgaattg 90360 tcttcatctc aacaatcctt cctattttgg gggtggcata tcctggtttc ccacagggtc 90420 ttgttacgaa gcagatattg aggtagtgga tctggggcaa ggcctgagag ctcccaggcg 90480 attcggacac tgttggtcca gggctgtgct gtgagaaaca gggttctgag cagctgtctc 90540 tgtgaggaag catgctgggt gtgcctcctt catcattgtc accccagacc ccagctcagc 90600 atctgaccca gggcagatgg tccatcaata tttgttaaat gagagaaaaa aaagtataca 90660 aacacttcaa cacacctcta ccttgtctat gcctttgctt gggagatgat gacggggcct 90720 catcagagcc acgggcacgt cctccagcca cttcctgtcc ccgctgtgga acctcacctt 90780 ttaaatctgt ccccatgttg cagactcccc tatgctctga ctgtatttat ttaaaacttc 90840 ccagtcccca tttaaatcct atgatgttct gtgactagca ttgtctttga cgtgagaaca 90900 tacaatgctt ttgtagcctc tgttatatga tatcaagctt catggtgaat aatctcactt 90960 agaaccaatc aataaaagaa tgcaagaacc tgagcagctc tcctcactag gatggccagt 91020 gtgctggcgg gcttcgctga agtgggattt ttcttttcac cattattttt ataaccacat 91080 tcaagggaaa ttctatttgg tgttaaaagt gatacttcac caagagtcaa aagagaaaac 91140 acagaaggct aaattctaga cttttttcag ctagcagcca gaggtcaaat tatttttcta 91200 agacccctgg tggacaaagc atttaatgat ctggatcaag gtaaataaaa tacgtttctc 91260 tggccgggcg cggtggctca tgcctataat cccagcactt tgggaagccg aggggggcgg 91320 atcacaaggt caggagttca agaccagcct ggccaatatg gtgaaacccc atctctacta 91380 aaaatacaaa aaaaaaaaaa atttagccgg gtgtggtggc gggtgcctgt agtcccagct 91440 actcgggagg ctgaggcagg agaatgccat gaacccagga ggtggagctt gcagtgagcc 91500 gagatcacac cactgcactc cagcctgggt gacagagcga gactccattt caaaaaaaat 91560 aaaaaaaata aaatatatat atatatatgt atatgtatgt atatatgtgt gtgtgtgtgt 91620 gtatgtgtgt nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 93000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 93060 nnnnnnnnnn nnnnnnnnnn nnnttgctac cctgaaaata tatatatata tgggctctgt 93120 cttctactag taacaaataa aaggtagact gaattcttgt atattcaatt cttatatatt 93180 cactttcaag ttaaggtgat attttttctc tctctggtga aaatacctgt cctagccatt 93240 gccaaaaagg acacactatt ttaaattctc ctttggtgct ggaaaaatct gagtcattta 93300 ccccctacta gatttcagga aacaggaagt ataaaattgc acatttaatt tgctacacac 93360 ttcaaacttt aggagatgca agactcatgt agtaaaagct gtgcaactca aacatccaat 93420 gggtagcttc tccccacaaa ctgtaatttt tgttggggca cattagggta tccagggaac 93480 tgctctgagc aagggggagg gaggtccagg tgtacttgta gcatatctgg ggaattctgt 93540 gtaagtatca gtcaccccca acctttaagg ttgccccatt tcacatctgt tatgatgaag 93600 gctttcggct gggggactgt ggttctggct gtagagtcag aatgaaagga acagctgaat 93660 atccctgttc atgtcagcat ttcagggcag aggatgagtc aagcacgatg tttactgagc 93720 agagccagag gcaagagcca cttttttctt gcagctctaa gaattttttg ttgctttttt 93780 tgtatgcttt gtttctcata catcagcatc atcatgcgtt gcacatacac attgtacata 93840 acaactgctt acagcatgcc gggcactgta ctaagccctc tgcaggtgat gactcaatta 93900 ttgctcccag caaccctcta aggtatgcca tgttatgatc tccactttgc agagaaggaa 93960 acaagcaccg gttaagcaac ctgcccaagc tagtatgtgt cagggtcagg accccagccc 94020 aggcagctgg tcccatgatc tgtgctcttg acttgcgtat attaacattt aatataatcc 94080 actatttaat atgtatgtag tgctgaatta aacaagagtg tgtcatttag gaaatggtga 94140 tcaaatcagt tcacgatccg atgaatactc aatgaatgac taccatgtgt atttccttaa 94200 atatactcaa cacttcccac cttccccatc agggtcctca cccagcaatc catgtgggga 94260 aaggggcaca aagttggccc acagtgggca ccgaatagat gcctgtttaa caaataaaag 94320 aatgggcaac tggaagagaa tttgtaggat attcacagaa tcaccaaatc cagtaattat 94380 ttcagtctag cattgcagtg gcacagaggc agtttttaag tttgtgaatg gctcttatgt 94440 aaaagatgtg gctatttgag gatgaaacaa gacaaaatgt ggtgaatgta cttcagccta 94500 agtgttttgg gggagggaga attgagaaaa atctctaggt ctaaacgctg gaacggatga 94560 ctgataaagt tagaatctct cttaaccagc tcattctaag tgtgtgagca tccgcattaa 94620 acctaatcta tggcctgata cttaaaaata acctgttata aagtaaccat aaaaaaactc 94680 acagatttca gcctgattca tttggccaca taaactacaa tcagcattaa tagcttccgt 94740 cagcatttat gccgtacttt ctattagcac gatgggttct acaatcattt atgagtttct 94800 ggaaagtcta atagaaaaca cacctacatg ttctctgtgg aaaatacaca ataggttttt 94860 ggggagtttt ttgtttgttt gtttgtttgc tcagggataa gccagaaggt gtcgccactg 94920 ccccatggct aaagtctggg ccgggttccc taaccagaac ttctcccctg atcactcctg 94980 agctcgctag ctctgcagct tcctcccaca ttcagtcagt ctctctctct ctctnnnnnn 95040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnttttaa acatagccaa atactggcca 95100 aagtctcttg cctttaaaaa aaatatgcat tttgttgttg tactaatact tctgaacata 95160 cgccaaatat aagggtgatt tgtggagctg tattaaataa aaagatgctt ctttataaga 95220 atcccctata gagcttgggg aagcagctat gtggagaatc tgtgtcttcc ttaagaaagt 95280 ttctgaattc cttggaaata catcagaaaa caccccattc tccccaccac agaggagggt 95340 gggcaaccaa cagtgtccta tatcccaacc caccataatt gtaagactta aacgctatct 95400 ttcaaataaa gaatcattta ctggcttctt actggaaaga actataggag cttgtcattt 95460 aattcatcct cccctctgag gctgaaattc tttctagaac aaacaaataa tacccatggc 95520 accacgtgct gcacactctc tgtagcccag ctgacgccac acgagggaac agtggggaat 95580 ctgttgaagt tgccattgca ttttgctttc tcataaaccg ctgtttgtct ttctgccttt 95640 cctgttttta gcagtgccta gaaacggagg cctgggaatt gcctccccca tgtgagcccc 95700 tgggttgttc acacccaagg ccaatttgac aacatcctcc tactctggct atgaggccaa 95760 gtccagtgtt ccttggttca gtgccaaagg tggacatgaa ctctctaaac tgtcaaacta 95820 agaaaatgtg acacacccat gtggcctgga gagagcaaag ccaaacccaa agagatgctt 95880 accaggcaac tttagatccc taacatgtta ggaaagtcaa gagcaagctt catcctgcaa 95940 gctctccagg attctccttg gacctggaac atgctaggat gttcagataa acttcacttc 96000 agacactgaa tttctgcctt gagctatctc ttctctcatc tttctctcgg tgtcttcttc 96060 cccttcatcc tctcataaac ccaagttcta tagctgtaat aataataata gcagttaact 96120 ttaatcaagt ctcactctgc accagacact agatgatgtg aacctgtgaa caagcataac 96180 tgaggattca aacccaaatc tcttatggca gagtgaatgt gctgctctgc tactgtgttt 96240 gttatcagaa gccatggctc cccgacatac tcgacccaga ttctctgttt cttccttgat 96300 gaaaccagtg acatgcaaac atagccagag ggcccaggca cagcagagag cgtcccatca 96360 gacctgatag aaagtcccct tcccatcttc taaaaccttg ctgccatttc tgacttcatg 96420 tggtctaaga atctggtgta gacattattt aaaagacaaa gggacctgaa aatcagcccc 96480 cccttacctg ccatgagtcc tagaaaggtc actgcggatg agaaaacccc agtttccacc 96540 tgctttataa ttttgccttt gtgtggtcat tcatactcat ataacctaac tgtaactggg 96600 ctactgttct ctttctttaa ggactttggc aactgagaaa aaagctacat gactcatttc 96660 taaaacctga aagatgaatg cagatcttta tgtaaagttt tctggtgggg gaggggattg 96720 gttacactgc ccatcacaga gctgaacatc ctcaaaatcc ccttagaata tctttgcctc 96780 tcctcaaaat tgagctcgtt atagcagcat ctacatttct gcctgcttgg cctgcacctg 96840 cccttttcct caccctccca atcctcctgg gccctgctgg ggctcctaca tcccgcttgc 96900 tcctggatct ttggcctagt ccatctgatc tctctcagtg tctcttcctc ttgtcctagc 96960 tgcctgtgga aagaaaggct tcttcttcca acagatgagt aagtggtgat attcttctta 97020 ttattatttt ttgtttccat cacgttacca ttcacgtttt aaactatcaa atgtcaacaa 97080 gtgtggccat ctttgtattt tctcttcaaa aatatgtgat ggccagagac atctccgtct 97140 gccatggtct ttctcaaacc accactgtac tttttttgca ccctttggtt atatcaacat 97200 gatctcaaat gagaagatga aaagtaaaaa cttcattcag tgctccaggt atttctcaat 97260 atctagatat ttctttcctc ctaggaaaaa tccaaatgga tttctcactt tttgaccaga 97320 tgtttcattg tcacacagcc cgtgtgacag cattggcccc tagtacaggc agagttgaaa 97380 agtgatgtga tgggtcatgg ttgcccctct actcagagaa aactccttct tgacctcatc 97440 acggtcgctg aaagtactaa gaattatatc tgaaatcaag cactggtagc agaacacaaa 97500 tatgttacca aattagttgg aaggtaaggc atgggggagt catgagggga gccccatcct 97560 cttttgtcag gggaatgaga acattttgca gcacctgtca ggccacgtga atacccaagg 97620 agtgatggga cagtccttgc acccagaagt ggccttgcag agtagaagga gtttatgttt 97680 tagaatctaa tagccctgcg cttaaatcct gggctttgca cctaaattat aatagagcta 97740 atcgattgaa catttttttc tctctgtcac tctgagtacg tgattctaat ctgccctagg 97800 cattcattga cctcagagag attaggattt ctgtaaaagg aactctaaga cagaaaaaag 97860 aattcaatat tcagcttttg atcttcaaaa tagcattttc aacaagacag tagactttta 97920 cccaacctat tgtctaagat gttctaaact cttttatttc tcaattttta cccatcaaaa 97980 accctgcctg gaggcctcga ctcacagagt agaaacagtt gtactggcta tgcagcaagg 98040 ccgcgaggct ttctgctcaa tgattggtgg gctatttgac ctgggaaaac taaaagaaaa 98100 gcccaccctt cctagcacag gggagttggg gatgaagaag aaaggagagg tggtgggggg 98160 gagggtcagg gaactagagc atgttaagag ctccgtgtgg tgctttgctt gggtatcctt 98220 ccagattgtg gagggaggca agcagcccac ctgtaacccc cacccagcat ccccatggtt 98280 actctggaag gaagcaaaca gaagaaatgc agaacctgta ttcgctttcc aaagccttcc 98340 acccatagat tccagaaacc ccatctcccc agtgctgtga ggtcacaggg gcctagtccc 98400 caattcccag tgccactgac tcaggaagga gagcaaggag gggcaggttc tctgggaaac 98460 tgagcatatc aatgcaaaga gacagaattc tctaggggga ctatttagat gattacatca 98520 gactaggtta taatccaaaa tggacgaaat taaagttgtt gttgtttttc caccacccag 98580 aggttaggag cttgtaagga agaccatagc agttgaaaac aaagcaaggc acatttccat 98640 tgtacgtccc aggagtagtt tgaatggttt aaggaccagc tatgtgtgtg cttgcataag 98700 cgtatgtgcc ctgggtaagt ctaagactca gtttcttcat ctgttgaatg acgatgaata 98760 atattttcca tctcaatagc ctcattgtgt gtttaaatgg tagttgcata aacatcccta 98820 gctcactgcg ggaattcagt gagtgtattt ccttgctggt gctaaggtat gggactaggg 98880 caataaaagg aacaagatag ggcagagggt actcggccca ggccagtgcc caataaccac 98940 aacctccttc tccatggaga gttgaacaga cacccatcca atctacagac aacggtcaca 99000 ggtgggttcc tggtttttat tttgctgcaa cacacataca agagaaaaac catatatgag 99060 aagttgagcg tgcagccacc tgggcccaca gagctgttct cattctttca aagaccacct 99120 ttgatatctg ctcctctcca tgtgaatttt gagccccagg agtctgcttt gccttatgct 99180 catcaggacc tgtgctgcac tgagtttgcc agcaaaccag ggctgagctg ttcttgcaag 99240 aacttgttgg aggctgggtt agagtcttgg ctccccagtc cccattcagc ctccatgccc 99300 tggtaagaat atattatttt cttagctcaa attgctgttt ccaagtcatt atttctctac 99360 gacgagaaaa caatattggg tcttatgtaa tatcaacctc aatgaatctg gaagtgatca 99420 tggaatgggt ggattttttt tcctccttaa aaaagagaaa aataaatcta agattgtttg 99480 catatggctc agaatattta tgaactattc cccttgtttc ctcatcacat attttaccca 99540 gtgcatttgt gtgtggtaca gaataaatct gtatggggag cagaggcgga gaggggaaga 99600 aagcagttaa aacactgggt atctatcaag tccctgggat gttagtgatc ctgcgtcttg 99660 ggtgccccaa ccctaggctc acttgctgta gttggaggga tacccccatc tagctactca 99720 gagtaattat gtggagtgag gatttcaaat atttacgagg aattaaacac aaagaattaa 99780 catctttttg ttgctctagt ttctttcaca tcccccttgt tcctaccaga gcagaaagtt 99840 caaatagttt catgacctgt gtaccatcag tcaaaagaga tcattaaatt ctatggatgg 99900 cagattgagc cagggtcaga ggaatgagtt ggcaggtggg aggaaaagaa ttggcagagc 99960 gtatagtggg tactgccatt tgctatccga taatatggta gtgtttcagc acacaggcat 100020 gttgtgtcta ttttgctatg ggtgtgcctt tcaacacagc ttatcaccaa ggagcatccc 100080 aagggagtat aaggaacagg gctttggctg tatactaaat aagtaaggga agcaaatctc 100140 caggctattg attttcctct ccagggagtt ggccacaaat ttggagcttc ttaaaatcac 100200 ccattctcca ctgcaccttg ggatattcag gaatatattg accaagagta tggggcaagg 100260 aacagagaac aggaggaccc cacgtttggt aaggcaatag caacaactac cattaattga 100320 tgtcatcgta ttggccaggc actgtattag gtgctttccc tgcattatct gagttaaccc 100380 cagcagtggt cttaacaggg tgaaggatcc ttagtcccgt tattgagatg agaaagcaga 100440 aactcagtga agtagaggaa cttgtccaag gtcacatgta agtgccagga tcaacgacat 100500 ttgtgggatt tatgaagcct taaggcaata caataagtac caatacagaa ttcttgaaat 100560 cagatccaag cgcttggaag ggacccagga aagcaagaca ccttgaagct taaggctcag 100620 cagcttccca gtgaattcac cctgggcggc cttgagattt aaacacagtt tagtcccagg 100680 ccgtgcagtc ctttccctgt tgccttgtct cctcttcctc taccacggct gttaccaact 100740 gcagccctca cattcctggt caaaaataca caaacccaag tgaggagact gatacacaga 100800 gggtgccttc cagaatgtgc tgacttggaa agctccatgg aatgtcacag ttgcaacatg 100860 aatcaacaat gattcctgtc accagacttg atgccttcca tctcgaggaa ttaccggagg 100920 gatataaaga actatcttcc caaggaactt caatcactgc tctaggcttt tcaccaaatt 100980 tcatgatatt tcccagacct ggtgacatgg gcacagactt gtgcaacagc ccactcaggt 101040 ctccagggcc atttgagatt agaacctgca atgtctcatc tcctatcgta tgactctttc 101100 cctcagatga tattagttgg tcaaattttg acaggcccag tatttgattc ttcctttctt 101160 cattataatc aagattttgc tgtaatctcc aggaatgaaa ggggtagagg gattgagcaa 101220 gcaaacaaat aaatcacaag tgcttcaggg agaaaagcat aataagcatt gtttagtacc 101280 caccctctgc cattgccttt gaatagcctt gcaatcccat cacagtctag acaattatta 101340 aggaatatac tgcattttcc cagccctgct tactctgttc tcccaaactg gaaacacaaa 101400 gcagatgatg aagtagttca aatgccacac cagggagaag aaagtattga gaaactatgc 101460 ccaacctcca ggcagttttc aaacattcca gtaagtgttc aattgattta tcttgggctg 101520 gtgtctttcc attgtctcaa acaaaaggcc ttctatgaaa agacaaaaag tgggttatga 101580 gaggagtggc taacaaggca cacttgggaa aatctgaaca agagtacagc cctcttggct 101640 ttgcctccag aaggcagtgt ccgccttgta tctactggaa tgaacagcag gatcgacttc 101700 cattatctca gtgtcaggct ctaaaccaga gtctggggat ggggctgatg acacaggtgc 101760 agtgactgat gccctggtca gccatctcct ccatgtttgt cctttgagct caagtagaaa 101820 gtttctatgg tagtgttctc tgattagaaa ggactacgag attctgggca aaacgatttt 101880 cagtgatgat ggcttcccac agttgttctt ttgggtattt tgtctgcttt tgtgtctaaa 101940 taaattatgt gacagataat tatttgacca agactgtaaa agccttctcc catgttatgt 102000 tcctttgggt gttcattcct tgagtcataa tcatgacagc tttctgtgca gctgggggtt 102060 gcacatttgt ggtggagccc ttccatttga gacagcagtg atttttagaa caagcttccc 102120 tagagtaacc caagtgttct tccctggtac ccatcacttc tctgggggtg acatcaaaaa 102180 gggctgttct attccccctg tcaagtcccc ctgaaggcct tgtccccaac tcttcttggg 102240 ttgggttagc tatagttaat acttccacaa tatctgatgc atatctttgt cttggaactt 102300 ctatatagtt tatatttacc catttaatag ccttttcttc ctaaaaatgt aatatgtagg 102360 ttgatgtgga atattagcaa attatagata accaaaactt tttaaaaaaa tggctagtat 102420 tgnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 102480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 102540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 102600 nnnnnnnnnn nnnnnattct cttgcctcag cctcccgagt agctgggtac tacaggcgcg 102660 tgccaccatg cccagctaat ttttgtagtt ttagtagaga cggagtttca ccataatggc 102720 tagggtggtt tcgatctctt aacgtcatga tctgcccgcc tcggcctccc aaagtgctgg 102780 ggttacaggt gtgagccacc acacccagac tgtgtttcta tatttatgta tttttttcaa 102840 agcaaaagat tagctagtgt ttattgtttg gttagagttt gccttttcag cctatttagt 102900 acacttctac tgcattgtgt taatggtttc caggtattct gcagtgtgtg gggagctggc 102960 agtgtgatga aggggacagg attctagatg agctactcca ctctgaagac cctatgactg 103020 tatccactgg caagaagttt gaataaaggc ccttttactc tttcatgcca gctcttgaaa 103080 gaggcctaac caaggctcta aagaaattgg atgactacct gaacacccct ctaccagagg 103140 agattgacgc caacacttgt ggggaagaca aggggtcccg gcgcaagttc ctggatgggg 103200 atgagctgac cctggctgac tgcaatctgt tgcccaagct ccatgtggtc aaggtaagag 103260 agctctaccc acaggggcct gcaagatcca gctccatctt aggcccaggt cacctgtgtg 103320 gatgagtcaa ggacagtacc acctgttggt caagaacctg gaccctgaag tcaggtaata 103380 aggacccaag gtcaccctct gctgcttgtt ggctgtgtgg cctccttgag cttcagtttt 103440 catttataaa ataggcatat attgcttact tcaagtgttg gtggaagagt aaatacagcg 103500 tgaaagtgct tggcatattg tgggggctta atatgtgtaa tagtcgcaat tatcgttgtt 103560 gtatacagtc atatcactcc aaaggcctct tcctcatagg atttccctgg ctacacccct 103620 acagctctat taaatgtgcc ctcatatgca ttttttcttt gtgcacagac ccaccttctc 103680 acttcctcca gcaacttcct aaggtgagcc cacattattt tcctcatcta tcaaatgaag 103740 aggtggaggt tgcaagaagt gatgtcactt tcttgctatc attgcactta ctaaccattt 103800 gcagcatgta gtgtcatcct ctcctatata acaaaccctg ggaatctgag agttggaaag 103860 gacatttaga ggtcatccaa aacaatctcc cacttcaacc tggaccactt tctgctgttt 103920 ctgtgacagg cttctgtgaa gctgtgactg cagcctctga gaacgaggag ccctctgctt 103980 aatgagccag ccctaccatg ttagatagct ctgattatta aatcattgtt ctttacaatg 104040 agcccaagca tgcctccctg ctatccattt tttctctaga gtaacagaga acagctttgc 104100 ttgccttcac ctcatttgaa gacagtagtt gtatccccct aagctctgtc aatgagcact 104160 tcttccccca ttctttcctg accctcgtca gcctagtatc agatagccat actgtgctct 104220 attttacgca tgcttatatc ttactgtctc agccagacag caaactctgt gaggaaagga 104280 actttttaaa gtgtgatggt gggcacacag tggccattca ataaatactc attgattgat 104340 catttgatca cctggtgtaa gtctttacga tatccagttt atttctatgc ttcactgaga 104400 agactcagat tcaatcatct gtcagctgag tatatgccta ttatttttca gctcaagtcc 104460 cgagtcaaaa atgctatctc ctctccaacc agagcatgta ccagcttgag ctgaagtcac 104520 tcagttgtgt gtacactggt gtttccgtat gccttagagc tgaagactga gagggaatca 104580 tgcataaaaa tggagtgggc aaatacaacc tatttagaaa agaacatttt ggatttgggg 104640 ccaagccaat agtcacttgt aggtccagcc aatctatgtc tctttgaagt tattaactac 104700 tgcatgcccc acccatcatc ctttattctt cttctcctta ggaacaagta ccactgaaag 104760 ggatgatata attccagctc agtcacactg tgtcagagtg atacaatgca aagatcagga 104820 gacccgagtt ccggtcctgt atttgctgcc aactagcagc atgagctgag gcacatcatt 104880 taatcttttt ggaattcatt tttctcgtgc ctagaagaac agaagtggat tgtattcctt 104940 cttgccttct tttcctttct tctttccctc cttctttcct tttctcttgc tcaaacatgt 105000 attcactacc actcaaaaac catttgttga acaaagcaaa caaatgaatc tcccaagcct 105060 tgggcttcat cctgtgattt cctcaattcc cacctgcctt aaattactca gtgaagccct 105120 gtccttggag aaaattcagt gggtggttaa cccagagaag ctggagatca aaaagaagat 105180 ggccaatgaa agaacaaagg ccagcccttg gcccctatct ctttggattt ctgctgatcc 105240 agcttatcag atcccagaaa cctggcaaac ctctaaagtt cacaaagagc gaaggggaag 105300 ccaagtcagg cctccagttt ggcttcggat gccaaaactt aatctgggct gtgggagcta 105360 actgttttca tatgaaagag caaattcaga acatgagcat ggaagtccct gcgaacgtca 105420 gatctccgtg tgcatcctta cccccttgct gctttcatgc tcactctcct cttgcgtggc 105480 tcgctttcag gtttatctcc atccctggaa gcagagttgc tctggcccag gctctccatg 105540 agagtttggc ttgaacattc attgtctggc cccctcctag ttctcatctc ccaaagtcaa 105600 gccaatgtgt gaagaaatga ccagctcagc agccaaggcc cagggtgcac aggtcttcgt 105660 tgggagaggc atctgcaggc ctttccttgc ccactgggat ccttgcctag catagtgacg 105720 atgttcagcc ctggagacaa acaagaaggg gaacaccaac atcaatagaa gtatatattt 105780 acaaattgca tttctgctgt attgaaacta acattctgcc ctttaaaatc ctgaaaataa 105840 aatttcagta tgaaatgact tgaggctact ctatgaatca gtgtgtcact gtgaaaaata 105900 cttttggatc cctttatctt attggagacc cttttcatcc actctgataa attccagcca 105960 gttctcttgg tcaggccacc actcctgcat gaatttgctc ttagccaaga cagcctcttc 106020 tcaaaggaac ttggcccaac ccaagggatc atcatctttc agtgaacaga aagggactgg 106080 ggagatatcg tggtggcatc tctcattgtg agagctttat caaaggactc ggacttcatc 106140 acccttgctt gtagttacct aagccagaca gaacagtgtg ggggtggctt ctttggtgcc 106200 cacaccaaac cagttatttt aacagagaga atttaaggaa gtactatgta ctaaagaact 106260 ggaaaggcaa aatatactag gaggttctac cttcaagagg cagctactac tcctagggca 106320 gga 106323 4 197 PRT Human 4 Met Thr Asp Ser Ala Thr Ala Asn Gly Asp Asp Ser Asp Pro Glu Ile 1 5 10 15 Glu Leu Phe Val Lys Ala Gly Ile Asp Gly Glu Ser Ile Gly Asn Cys 20 25 30 Pro Phe Ser Gln Arg Leu Phe Met Ile Leu Trp Leu Lys Gly Val Val 35 40 45 Phe Asn Val Thr Thr Val Asp Leu Lys Arg Lys Pro Ala Asp Leu His 50 55 60 Asn Leu Ala Pro Gly Thr His Pro Pro Phe Leu Thr Phe Asn Gly Asp 65 70 75 80 Val Lys Thr Asp Val Asn Lys Ile Glu Glu Phe Leu Glu Glu Thr Leu 85 90 95 Thr Pro Glu Lys Tyr Pro Lys Leu Ala Ala Lys His Arg Glu Ser Asn 100 105 110 Thr Ala Gly Ile Asp Ile Phe Ser Lys Phe Ser Ala Tyr Ile Lys Asn 115 120 125 Thr Lys Gln Gln Asn Asn Ala Ala Leu Glu Arg Gly Leu Thr Lys Ala 130 135 140 Leu Lys Lys Leu Asp Asp Tyr Leu Asn Thr Pro Leu Pro Glu Glu Ile 145 150 155 160 Asp Ala Asn Thr Cys Gly Glu Asp Lys Gly Ser Arg Arg Lys Phe Leu 165 170 175 Asp Gly Asp Glu Leu Thr Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu 180 185 190 His Val Val Lys Ile 195 5 197 PRT Rattus norvegicus 5 Met Thr Asp Ser Ala Thr Ala Asn Gly Asp Asp Arg Asp Pro Glu Ile 1 5 10 15 Glu Leu Phe Val Lys Ala Gly Ile Asp Gly Glu Ser Ile Gly Asn Cys 20 25 30 Pro Phe Ser Gln Arg Leu Phe Met Ile Leu Trp Leu Lys Gly Val Val 35 40 45 Phe Asn Val Thr Thr Val Asp Leu Lys Arg Lys Pro Ala Asp Leu His 50 55 60 Asn Leu Ala Pro Gly Thr His Pro Pro Phe Leu Thr Phe Asn Gly Asp 65 70 75 80 Val Lys Thr Asp Val Asn Lys Ile Glu Glu Phe Leu Glu Glu Thr Leu 85 90 95 Thr Pro Glu Lys Tyr Pro Lys Leu Ala Ala Arg His Arg Glu Ser Asn 100 105 110 Thr Ala Gly Ile Asp Ile Phe Ser Lys Phe Ser Ala Tyr Ile Lys Asn 115 120 125 Thr Lys Gln Gln Asn Asn Ala Ala Leu Glu Arg Gly Leu Thr Lys Ala 130 135 140 Leu Arg Lys Leu Asp Asp Tyr Leu Asn Thr Pro Leu Pro Glu Glu Ile 145 150 155 160 Asp Thr Asn Thr His Gly Asp Glu Lys Gly Ser Gln Arg Lys Phe Leu 165 170 175 Asp Gly Asp Glu Leu Thr Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu 180 185 190 His Val Val Lys Ile 195 

That which is claimed is:
 1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO: 2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO: 2, wherein said fragment comprises at least 10 contiguous amino acids.
 2. An isolated peptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO: 2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acid sequence shown in SEQ ID NO: 2, wherein said fragment comprises at least 10 contiguous amino acids.
 3. An isolated antibody that selectively binds to a peptide of claim
 2. 4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO: 2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO: 2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).
 5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO: 2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or3; (d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO: 2, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acid molecule of claim
 5. 7. A transgenic non-human animal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising a nucleic acid molecule of claim
 5. 9. A host cell containing the vector of claim
 8. 10. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 11. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 12. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.
 13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample.
 14. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.
 15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.
 16. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.
 17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor.
 18. A method for treating a disease or condition mediated by a human transporter protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim
 16. 19. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.
 20. An isolated human transporter peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence shown in SEQ ID NO:
 2. 21. A peptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence shown in SEQ ID NO:
 2. 22. An isolated nucleic acid molecule encoding a human transporter peptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule shown in SEQ ID NOS: 1 or
 3. 23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule shown in SEQ ID NOS: 1 or
 3. 